Methods of cancer diagnosis and therapy targeted against a cancer stem line

ABSTRACT

Improved methods for treatment of cancer which involve the targeting of slow-growing, relatively mutationally-spared cancer stem line are provided. These methods are an improvement over previous cancer therapeutic methods because they provide for very early cancer treatment and reduce the likelihood of clinical relapse after treatment.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 09/468,286 filed on Dec. 20, 1999, now U.S. Pat.No. 7,361,336, which is a continuation-in-part of U.S. patentapplication Ser. No. 08/933,330, filed Sep. 18, 1997, now U.S. Pat. No.6,004,528, issued Dec. 21, 1999, each of which is incorporated byreference in its entirety herein.

FIELD OF THE INVENTION

The present invention relates to novel methods for the diagnosis andtreatment of cancer, and is based on the novel OSES (“one-stepepigenetic switch”) model of carcinogenesis.

BACKGROUND OF THE INVENTION

Classical cancer models to date which have attempted to explain thecharacter of cancer cells have typically described them as fast-growingand highly mutant cells. These cancer cells are hypothesized to havebeen produced during carcinogenesis because of a multi-stepneo-Darwinian evolutionary process involving mutation-selection eventsat the cellular level (Fearon et al, “A Genetic Model for ColorectalTumorigenesis”, Cell, 61:759-767 (1990); Nowell, “The Clonal Evolutionof Tumor Cell Populations”, Science (Washington, D.C.), 194:23-28(1976)).

Related to this, conventional cancer diagnoses and therapies to datehave attempted to selectively detect and eradicate neoplastic cells thatare largely fast-growing and mutant. For example, conventional cancerchemotherapies (e.g., alkylating agents such as cyclophosphamide,anti-metabolites such as 5-Fluorouracil, plant alkaloids such asvincristne) in a similar manner to conventional irradiation therapiesboth exert their toxic effects on cancer cells by interfering withnumerous cellular mechanisms involved in cell growth and DNAreplication. Other less commonly used experimental cancer therapies haveincluded use of immunotherapies wherein the administration oftherapeutic agents which selectively bind mutant tumor antigens onfast-growing cancer cells (e.g., monoclonal antibodies) have beenattached to therapeutic moieties such as toxins, radionuclides orchemotherapeutic agents for the purpose of eradicating fast-growingmutant cancer cells. Similarly, newer experimental gene-directedtherapies have attempted to exploit certain cancer-related mutations bycorrecting or replacing such defects by, e.g., inserting wildtypeversions of such mutant genes (e.g., p53) into cancer cells, inhibitingoveractive oncogenes (e.g., ras) in tumor cells, or by using existingmutations in cancer cells as targets for other therapeutic (e.g., viral)vectors (Feng et al, “Neoplastic reversion accomplished by highefficiency adenoviral-mediated delivery of an anti-ras ribozyme”, CancerRes., 55:2024-2028 (1995); Bischoff et al, “An adenovirus thatreplicates in p53-deficient human tumor cells”, Science, 274:373-376(1997)). While all of these methods (i.e., conventional chemotherapies,irradiation, immunotherapies, and gene therapies) may, by their design,eradicate a significant proportion of a given tumor mass by destroyingthe large populations of highly proliferative and mutant neoplasticcells thus resulting in a clinical remission, in time the tumor mayrecur at the same or different site(s), thereby indicating that not allcancer cells have been eradicated by these methods. A number of reasonsfor tumor relapse have been offered within the conventional paradigm.These include insufficient chemotherapeutic dosage (limited by onset ofsignificant side effects), and/or emergence of cancer clones which areresistant to therapy.

The novel model for carcinogenesis presented here (termed the OSESmodel) offers an alternative explanation for relapse wherein (as will bediscussed in more detail) a clandestinae slow-growing relativelymutationally-spared cancer stem line acts as the immortal founder lineof a tumor and produces as its progeny the highly proliferative mutantcancer cell populations targeted by conventional therapies mentionedabove. Accordingly, it will be shown that this cancer stem line(hypothesized to exist by the OSES model) is not targeted byconventionally-based therapies (designed to target fast-growing largelymutant cells rather than slow-growing non-mutant cells). In this manner,the untargeted cancer stem line can gradually regrow the tumor massfollowing standard therapy thereby leading to treatment failure andclinical relapse.

Recently much evidence has been accumulated which raises significantconcerns as to the validity of classical cancer models based on theneo-Darwinian paradigm—and thus also to the efficacies of cancertherapies wholly based on this model. So that the invention may beunderstood, previous conventional cancer models and their inadequaciesare discussed below.

There is extensive evidence that cancer results from the evolution of anincreasingly “cancer-like” tissue type leading ultimately to one withmalignant capability (Furth, “Conditioned and autonomous neoplasms: areview”, Cancer Res., 13:477-492 (1953); Foulds, “The natural history ofcancer”, J. Chronic Dis., 8:2-37 (1958)). That mutagenesis is causallyinvolved in the initiation of this process is a concept which hasstemmed largely from demonstrations that genotoxic carcinogens can causecellular transformation and that this “initiated” phenotype is rare,permanent, focal and heritable. (Berenblum, “Sequential aspects ofchemical carcinogenesis: skin, in: F. F. Becker (ed.), Cancer: aComprehensive Review, pp. 451-484, New York, Plenum Publishing Corp.(1982); Cohen et al, “Genetic Errors, Cell Proliferation, andCarcinogenesis”, Cancer Res., 51:6493-6505 (1991)). Moreover, that amutant gene can bestow a transmissible cancer phenotype is evidenced bytransfection and transgenic experiments as well as recent geneticlinkage analyses of human familial cancers (Weinberg, “Oncogenes,Anti-oncogenes, and the Molecular Basis of Multistep Carcinogenesis”,Cancer Res., 49: 3713-3721 (1989); Tsukamoto et al, “Expression of theint-1 gene in transgenic mice is associated with mammary glandhyperplasia and adenocarcinomas in male and female mice”, Cell, 55:619-625 (1988); Knudson, “Hereditary cancer, oncogenes, andantioncogenes”, Cancer Res., 45: 1437-1343 (1985)). It is also clearthat while the onset of carcinogenesis requires an initial (acquired orinherited) mutagenic insult, subsequent alterations are also necessaryfor attainment of malignancy (Nowell; “The Clonal Evolution of TumorCell Populations.”, Science (Washington D.C.), 194: 23-28 (1976)).

This concept is supported by findings that certain cultured cellstransfected with a single oncogene require additional alterations tobecome fully transformed (Weinberg, “Oncogenes, Anti-oncogenes, and theMolecular Basis of Multistep Carcinogenesis”, Cancer Res., 49:3713-3721(1989)). Moreover, epidemiological data implicating a series of ratelimiting steps in the pathogenesis of human cancers has lent support tothe concept that a gradual cellular progression toward increasingmalignancy is driven by neo-Darwinian mutation-selection (Miller, “Onthe Nature of Susceptibility to Cancer”, Cancer, 46:1307-1318 (1980).

Also, the well-documented association between increasing mutational loadand tumor grade has led to the seemingly most parsimonious mechanisticexplanation of these data whereby mutations cause both the initiation aswell as progression of neoplasia via a continuum of mutation—selectionevents at the cellular level (Nowell, “The Clonal Evolution of TumorCell Populations.”, Science (Washington D.C.), 194: 23-28 (1976); Fearonet al, “A Genetic Model for Colorectal Tumorigenesis”, Cell, 61: 759-767(1990)). Additional support for this neo-Darwinian model derives fromrecent high-resolution molecular analyses of human tumor biopsyspecimens that reveal a tight correlation between the appearance ofcertain defined genetic alterations and the transition to increasingly“cancerous-appearing” tumor regions (Fearon et al, “A Genetic Model forChlorectal Tumorigenesis”, Cell, 61: 759-767 (1990); Sidransky et al,“Clonal expansion of p53 mutant cells is associated with brain tumorprogression”, Nature (Lond.), 355: 846-847 (1992); Sato et al,“Allelotype of Breast Cancer: Cumulative Allele Losses Promote TumorProgression in Primary Breast Cancer”, Cancer Res., 50: 7184-7189(1990)).

However, it should be noted that there is a body of cancer literaturenot readily accounted for by standard neo-Darwinian mutation-selectionmodels. Namely, there are a host of independent data describingunexpectedly elevated transformation rates observed in certaincarcinogen-treated cells not fully accounted for by somatic mutationalone as well as the capability of some highly malignant tumor types todifferentiate or even revert to normal under certain conditions—findingsalso not readily explained by conventional mutation-selection models(Kennedy et al, “Timing of the steps in transformation of C3H 10T1/2cells by X-irradiation”, Nature (Lond.), 307: 85-86 (1984); Rubin,“Cancer as a Dynamic Developmental Disorder”, Cancer Res., 45: 2935-2942(1985); Farber et al, “Cellular Adaptation in the Origin and Developmentof Cancer”, Cancer Res., 51: 2751-2761 (1991)). Accordingly, alternativemodels to mutation-selection (e.g., invoking a role for potentiallyreversible non-mutational/epigenetic alterations as effectors ofcellular evolution toward increasing malignancy) have been advanced byseveral investigators in order to explain these and other relatedphenomena.

For example, as alluded to, while only a minority of mouse prostatecells exposed to methylcholanthrene initially become transformed, theentire population of treated cells rear progeny with an increasedpropensity for transformation at subsequent cell divisions despiteremoval of the carcinogenic agent. Similarly, treatment of variousrodent cells with other types of carcinogens, namely X-irradiation orretroviral infection, also results in transformation of progeny ofinitially untransformed cells at an overall rate-difficult to reconcileby somatic mutagenesis alone (Farber et al, “Cellular Adaptation in theOrigin and Development of Cancer”, Cancer Res., 51: 2751-2761 (1991)).In addition, Rubin has demonstrated that although only a small fractionof growth-constrained NIH3T3 cells form transformed foci, the entirepopulation of these murine fibroblasts gives rise to clones withelevated transformation rates (Rubin, “Cellular epigenetics: Control ofthe size, shape, and spatial distribution of transformed foci byinteractions between the transformed and nontransformed cells”, Proc.Natl. Acad. Sci. USA, 91: 6619-6623 (1994)). In a related manner, whileonly a minority of in vivo DMBA-treated murine skin cells becometransformed, heritable phenotypic alterations are present in the entirebasal skin cell layer exposed to this chemical carcinogen therebycorroborating those mentioned in vitro experiments cited in support of awidespread heritable phenotypic effect by certain carcinogens beyondthat which can be attributed wholly to their mutagenic effects (Farberet al, “Cellular Adaptation in the Origin and Development of Cancer”,Cancer Res., 51: 2751-2761 (1991)).

It has also been noted, as mentioned, that a variety of cancer celltypes can differentiate to varying degrees. For example, humanneuroblastoma cells sprout axons and dendrites when grown as explantsand murine leukemic cells differentiate into benign granulocytes andmacrophages when grown in vitro. Moreover, tritiated thymidine labelingof rodent squamous cell carcinomas and skeletal muscle tumorsillustrates that poorly differentiated cells within these tumors cangive rise to well-differentiated squamous epithelia and multinucleatedmyotubes, respectively. In addition, somatic tissues of transplantablemouse teratocarcinomas have been shown to be benign differentiatedprogeny of a subpopulation of poorly differentiated embryonal carcinomacells within these particular tumors. Most recently, all-trans-retinoicacid (ATRA) has been found to be efficacious in the treatment of humanacute promyelocytic leukemia (APL) by inducing terminal differentiationof malignant leukocytes (Pierce et al (eds.), “Cancer: a problem ofdevelopmental biology”, New Jersey: Prentice Hall Inc. (1978); Degos etal, “All Trans-Retinoic Acid as a Differentiating Agent in the Treatmentof Acute Promyelocytic Leukemia”, Blood, 85: 2643-2653 (1995)).

Accordingly, it had been suggested by some investigators thatconventional cancer models may not adequately explain elevatedtransformation rates of certain cells types (not adequately explained bysomatic mutation alone), or the differentiation capability of certaintumor cells presumably having arisen via a cascade of random geneticderangements. Based on these and other related observations, Rubin,Farber, and Pierce among others have theorized that the ability ofcertain cancer cells to differentiate might suggest that a cancer celloriginates from potent developing/renewing cells which undergo defectivemorphogenesis rather than from mutant cells which undergodedifferentiation. Moreover, it has been argued by several investigatorsthat mechanistically such defective morphogenesis is likely to arisefrom a series of non-mutational (i.e., epigenetic) alterations (ratherthan mutations) which by definition leave the genome fairly intact anddifferentiation-related genes functional. Cited in additional support ofthis cancer theory is the observation that certain malignant cellscannot only produce differentiated progeny but can also do so in arelatively orderly manner which mimics normal tissue development underspecified conditions. For example, neuroblastoma growth anddifferentiation is regulated when placed into neurula-stage embryos, andthe behavior of melanoma cells is controlled when transplanted intofetal skin (Podesta et al, “The neurula stage mouse embryo in control ofneuroblastoma”, Proc. Natl. Acad. Sci. USA, 81: 7608-7611 (1984);Gerschenson et al, “Regulation of melanoma by the embryonic skin”, Proc.Natl. Acad. Sci. USA, 83: 7307-7310 (1980)). In addition, leukemic cellsdifferentiate into normal hematopoietic tissue when injected into mouseembryos during leukocyte progenitor development and placement ofmalignant embryonal carcinoma cells into the embryonic milieu of adeveloping blastocyst results in complete reversion and differentiationof these cells into multiple tissue types leading to the formation ofviable murine chimeras (Pierce et al (eds.), “Cancer: a problem ofdevelopmental biology”, New Jersey: Prentice Hall Inc. (1978); Gootwineet al, “Participation of myeloid leukaemic cells injected into embryosin hematopoietic differentiation in adult mice”, Nature (London), 299:63-65 (1982)). In a related manner, restoration of native environmentalconditions at a particular adult tissue locale (i.e., withouttransplantation) causes certain tumors to “behave” as a normaltissue—e.g., resolution of normal physiologic hormonal balance causessome murine endocrine tumors to regress and certain rodent sarcomasinduced by implantation of an inert material into connective tissuesrevert when proper tissue architecture is restored (Rubin, “Cancer as aDynamic Developmental Disorder”, Cancer Res., 45: 2935-2942 (1985)).

That altered epigenesis can lead to neoplasia is an idea which is alsosupported a significant literature documenting the unexpectedreversibility of certain carcinogen-transformed cells in culture(Kennedy et al, “Timing of the steps in transformation of C3H 10T1/2cells by X-irradiation”, Nature (Lond.), 307: 85-86 (1984); Rubin,“Cancer as a Dynamic Developmental Disorder”, Cancer Res., 45: 2935-2942is (1985)). This idea is also supported by demonstrations thatapparently non-mutational events such as transplantation of normalrodent tissues (e.g., testis, pituitary, embryonic ectoderm) to ectopicsites can lead to their neoplastic transformation, while replacement ofsome of these cancer cell types back into their endogenousmicro-environments results in their reversion to normalcy (Rubin,“Cancer as a Dynamic Developmental Disorder”, Cancer Res., 45:2935-2942(1985); Farber and Rubin, “Cellular Adaptation in the Origin andDevelopment of Cancer”, Cancer Res., 51:2751-2761 (1991)). Also,evidence that some pre-malignant lesions may be reversible during theirearly stages has led to the proposal by Farber that a “pre-malignant”phenotype may be no more than a programmed adaptive (epigenetic)cellular response rather than an aberrant product of random permanentmutational events (Farber, “The Multistep Nature of Cancer Development”,Cancer Res., 44:4217-4223 (1984); Farber and Rubin, “Cellular Adaptationin the Origin and Development of Cancer”, Cancer Res., 51:2751-2761(1991)).

Accordingly, Rubin, a major proponent of the epigenetic model forcancer, had theorized that the evolution toward malignancy might be dueto the progressive selection (i.e., evolution) of increasingly“cancer-like” cells undergoing advantageous epigenetic fluctuations.Moreover, Rubin, as well as Prehn, have offered that the presence ofmutational alterations in tumors may not always have a causal role intumor progression, but rather may be the result of genomic instabilityassociated with the malignant phenotype thereby representing acancer-related “epiphenomenon”. (Rubin, “Cancer as a DynamicDevelopmental Disorder”, Cancer Res., 45:2935-2942 (1985); Prehn,“Cancers Begets Mutations versus Mutations Beget Cancers”, Cancer Res.,54:5296-5300 (1994).

However, considering increasing current molecular evidence in support ofa causal role for mutagenesis in human cancer, it is no wonder thatattempts have been made not only to consider alternative explanationsfor the action of epigenesis in carcinogenesis (i.e., other thanaberrant morphogenesis which has been linked to the unpopular notion ofa non-causal epiphenomenal role for mutations in cancer), but also moreradically to regard epigenesis—related data in general as largelyanecdotal. Notwithstanding, documentation that a variety of epigeneticalterations (such as changes in DNA methylation and genomic imprinting)are present in human biopsy specimens of a number of different tumortypes has undoubtedly helped to resurrect epigenesis as a realcancer-related entity rather than an experimental quirk (Goelz et al,“Hypomethylation of DNA from benign and malignant human colon“neoplasms”, Science (Washington, D.C.), 228: 187-190 (1985); Feinberg,“Genomic imprinting and gene activation in human cancer”, Nature Genet.,2: 110-113 (1993)). Accordingly, current cancer models largely of themutation-selection variety now regularly include aspects of alteredepigenesis into a more general neo-Darwinian paradigm wherein bothmutagenesis and epigenesis contribute to cancer evolution by acting asindependent effectors of cell variability (Vogelstein et al, “Themultistep nature of cancer”, Trends Genet., 9: 138-141 (1993)). Thus, incontrast to past models of epigenesis (which have regarded mutations asnon-causal), this combined paradigm is able to maintain the welldocumented causal role for mutagenesis in carcinogenesis.

However, it should be noted that while combining mutagenesis andepigenesis into one general paradigm has the obvious appeasing benefitsthat accompany compromise, it is still not obvious how such a combinedparadigm is any more apt than pure mutagenic models to explain instancesof tumor cell regulation, differentiation, or regression if it stillinvokes rare irreversible (genomic) derangements as major effectors ofcell variability. In reference to that model which best accounts forthese enigmatic data (i.e., the depiction of cancer as an aberrancy intissue morphogenesis), it is clear that its current state of relativeobscurity can be largely attributed to its unfortunate coupling to theill-fated notion that cancer-related mutations are mere epiphenomena—astance clearly at odds with recent data. However, by dismissing thisentire model because of an overzealous error in deducing itsconsequences, is it possible that some have effectively “thrown out thebaby with the bathwater”, so to speak? Alternatively, if one uncouplesthe concept of aberrant morphogenesis from any particular stance as tothe causality of mutagenesis in carcinogenesis, cancer could then beviewed in a new light as an epigenetic defect in tissue morphogenesisbut one which could also, in a seemingly contradictory manner, beabetted by mutations. For example, mutagenesis could cause cancer, notvia standard stepwise mutation-selection, but rather by triggering anactively developing/renewing adult tissue to undergo a largelyepigenetic-driven aberrant morphogenetic program. Moreover, subsequentto the birth of a cancer cell in this manner, mutagenesis could then actin another novel non-neo-Darwinian manner by blocking cancer cellreversion (i.e., rather than by promoting progression of pre-cancerousintermediates). Such a model would maintain the causal role ofmutagenesis in carcinogenesis while more readily accounting for theepigenetic nature of certain cancers than does the current combinedneo-Darwinian model. The preceding scenario is the basis for the OSESmodel for carcinogenesis, the specific mechanisms of which will bediscussed in more detail.

Accordingly, this report will contend that upon closer analysis of thecancer literature: 1) the data normally cited in favor ofmutation-selection are not exclusive to the conventional paradigm butrather are also consistent with an alternative and novelnon-neo-Darwinian model (termed the OSES model), and that 2) this novelOSES model may have an advantage in being more able than theconventional paradigm to account for certain past as well as morerecently described enigmatic cancer-related phenomena.

Thus, the present invention is based on a novel and improved model forcarcinogenesis which incorporates and indeed reconciles the presence ofthe seemingly conflicting processes of epigenesis and mutagenesis thatoccur during carcinogenesis. Moreover, based on this novel model ofcarcinogenesis (i.e., the OSES model), the present invention furtherprovides novel and improved methods for the diagnosis and treatment ofcancer. These novel methods should alleviate and potentially preventproblems associated with those of conventional cancer diagnosis andtherapy, in particular, the need for methods which provide for muchearlier cancer diagnosis than is currently available, as well as theneed for more successful initial therapies for cancer that are not assusceptible to tumor relapse as are conventional treatment regimens.

OBJECTS OF THE INVENTION

Thus, it is an object of the invention to provide a novel cancermodel—termed the OSES (one step-epigenetic switch) cancer model—whichclarifies and repairs the inadequacies of previous cancer models.

Moreover, it is a further object of the invention to provide novelmethods of treatment and diagnosis of cancer which are based on the OSESmodel.

It is a more specific object of the invention to provide a method ofcancer diagnosis which identifies slow-growing, relativelymutationally-spared symmetrically-dividing stem cells (i.e., a cancerstem line) which is the immortal founder line that rears those (largelymortal) highly proliferative mutant cancer cells normally targeted byconventional diagnostic methods.

It is a more specific object of the invention to provide a method ofcancer therapy which targets slow growing, relativelymutationally-spared symmetrically dividing stem cells (i.e., a cancerstem line) which is the immortal founder line that rears those (largelymortal) highly proliferative mutant cancer cells normally targeted byconventional therapies.

It is another specific object of the invention to provide novel andimproved cancer therapies which eradicate a cancer stem line therebydestroying the immortal portion of the tumor (i.e., the cancer stemline) and in doing so providing a true cure by preventing clinicalrelapse.

It is a more specific object of the invention to provide cancertherapies which target antigens present on the cancer stem line for thepurpose of destroying the cancer stem line.

It is another specific object of the invention to provide a novel methodof cancer therapy which induces, in a cancer stem line, a permanentswitch from symmetric to asymmetric mitosis.

It is still another specific object of the invention to provide a novelmethod of cancer therapy which induces, in a cancer stem line, terminaldifferentiation and/or programmed cell death.

It is still another specific object of the invention to spare normalstem cells of significant OSES-based therapy-induced toxicities.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides novel methods for the treatment anddetection of cancer which follow from the OSES model of carcinogenesis.In brief, the OSES model concludes that a clandestine relativelymutationally-spared immortal founder line (i.e., cancer stem line)exists within tumors and is responsible for fueling tumor immortality.Since the cancer stem line is directly derived from normal stem cells,as will be described, the cancer stem line (like a normal stem cell) isslow-growing and non-mutant and (like a normal stem cell) rears atransit population of highly proliferative progeny cells (which may bemutant in the case of cancer stem line progeny). Such highlyproliferative and largely mortal cancer stem line progeny make up thebulk of the resulting tumor mass (in an analogous manner to whichproliferative mortal progeny of normal stem cells make up the bulk of anormal developing tissue).

Essentially, while conventional cancer models invoke the presence ofhighly proliferative mutant cancers (hypothesized to be produced bystepwise neo-Darwinian mutation-selection), they have been largelyunaware of the OSES-proposed presence of an underlying slow-growingrelatively mutationally-spared immortal cancer stem line that rears suchproliferative mutant cells as its mortal progeny.

Moreover, this deficiency by conventional models explains many of theinadequacies of treatment regimens derived thereof, e.g., conventionalchemotherapies, irradiation, experimental immunotherapies, as well asnewer gene-directed therapies designed for treatment of cancer. Ingeneral, such conventionally-based methods attempt to eradicatefast-growing mutant cancer cells. This idea has clinical utility as, ifsuccessful, such methods may destroy the highly proliferative mutantprogeny of the cancer stem line and thereby diminish tumor burden (sincemortal cancer stem line progeny make up the bulk of the tumor mass),thus potentially effecting clinical remission (due to significantdecrease in tumor cell burden). However, a problem associated with suchtreatments is that the targeted highly proliferative mutant cancer cellsare largely mortal while their immortal progenitor, i.e., the cancerstem line, will remain spared of such therapies. This is disadvantageousas the cancer stem line over time can rear more highly proliferativemutant cancer cells, thereby effecting an increase in tumor cell burdenand clinical relapse.

By contrast, the subject invention provides novel therapies whicheradicate the slow-growing relatively mutationally-spared cancer stemline which is the progenitor of the larger population of highlyproliferative, largely mortal, often mutant cancer cells. Therefore, thepresent invention may provide a true cancer “cure” as it would eradicatethe founder line thereby alleviating and potentially preventing clinicalrelapse.

Also, the present invention provides novel methods of cancer diagnosisby early detection of cancer stem lines. This will be effected bydetecting factors (e.g., but not exclusive to, cell surface antigens, orintracellular factors) specific to cancer stem lines, e.g., via use ofcancer stem line-specific monoclonal antibodies attached to a readilydetectable moiety (e.g., fluorescent or radionuclide tag).

As noted, the subject cancer stem line, which can be considered to bethe progenitor of highly proliferative mutant cancer cells, is directlyderived from normal stem cells and as will be presented is functionallyequivalent to stem cells that have undergone an aberrant one-stepepigenetic switch (OSES) in mitotic mode from an asymmetric to asymmetric type. Therefore, the methods of therapy which arise from theOSES model of carcinogenesis will include, but are not exclusive to, thefollowing:

-   -   (i) specific cytotoxic targeting of the cancer stem line, e.g.,        via immunotherapy designed to cancer stem line-specific antigens        (some of which are expressed in and inherited en bloc from        normal stem cells;    -   (ii) induction of permanent switch, in the cancer stem line,        from symmetric back to asymmetric mitosis, e.g., via        activating/suppressing cellular factors which are involved in        this switch in normal stem cells; and/or    -   (iii) induction of permanent terminal differentiation and/or        apoptosis of the cancer stem line by forcing the cancer stem        line from a symmetric proliferative mitotic program to a        symmetric terminal differentiation/apoptotic program whereby        proliferative capacity is irreversibly lost.

DETAILED DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show mitotic of cell division modes (normal versuscancerous). Large circles represent individual stem cells, small circlesrepresent their differentiating progeny cells.

FIG. 1A shows normal asymmetric mitosis of stem cells. The immortal stemline in this case is the renewed normal stem cell while all of itsprogeny are mortal and destined to differentiate. This represents anarithmetic growth pattern.

FIG. 1B shows symmetric mitosis of stem cells (i.e., cancer). Theimmortal stem lines in this case are the cancer stem lines which fueltumor growth. This represents an exponential growth pattern. It shouldbe noted that, by the OSES model, stem cells which are dividingasymmetrically are intrinsically the same as those dividingsymmetrically, i.e., the mitotic mode by which stem cells divide isdetermined by their local surroundings. Thus asymmetrically dividingstem cells have normal surroundings while disrupted surroundings leadstem cells to divide symmetrically.

FIGS. 2A and 2B illustrate possible explanations for relapses thatfollow conventional chemotherapies. Large circles represent tumors,small circles within the large circles represent individual tumor cells;●=mutant cancer cells susceptible to therapy, ◯=cancer stem line cells;

mutant cancer cells resistant to therapy.

FIG. 2A illustrates the conventional model, wherein cells that survivestandard chemotherapy are speculated to have acquired mutations whichmake them resistant to therapy.

FIG. 2B illustrates the OSES model, wherein cells that survive standardchemotherapy are speculated to have either acquired mutations which makethem resistant, or may constitute a subpopulation of relativelymutationally-spared cells that are slow-growing (i.e., the cancer stemline) and are thus spared by therapies designed to attack fast-growingmutant cells. By this model, mutationally-resistant cells may have afinite life-span (having been initiated down a differentiation pathway)thereby making their presence following standard chemotherapy lessproblematic than the presence of an immortal cancer stem line. It shouldbe noted that the presence of cancer stem line cells resistant tostandard therapies is a novel OSES-based proposal not predicted byconventional models.

FIGS. 3A, 3B and 3C contain schematic of novel OSES-based cancertherapies. Large circles represent immortal cancer stem line cells,small circles represent mortal progeny cells of the cancer stem linethat are either differentiating or undergoing programmed cell death.

FIG. 3A illustrates that monoclonal antibody therapy to antigens presenton a cancer stem line leads to cytotoxicity of the cancer stem line.

FIG. 3B illustrates that certain. OSES-based therapies force a cancerstem line cell to undergo asymmetric mitosis (i.e., arithmetic growth).

FIG. 3C illustrates other OSES-based therapies force a cancer stem lineto undergo symmetric differentiation or programmed cell death(apoptosis) thereby extinguishing the cancer stem line and tumorimmortality.

FIG. 4 depicts the asymmetric mitotic pathway of a stem cell. Multipleligand-receptor binding action (left side of figure) leads to a signaltransduction pathway (arrow) characterized by induction of multipleintracellular activators and inhibitors of this pathway actingpositively and negatively, respectively (along branches on both sides offigure). This pathway ultimately leads to the unequal segregation offactors (right side of figure) to 2 qualitatively distinct daughtercells (i.e., asymmetric mitosis). Somewhere amid this pathway may existone or more “bottlenecks” (only one has been displayed for simplicity)wherein cellular decisions are made from a consensus of competingsignals. Such “bottlenecks” present convenient points at which totherapeutically intervene (for the purpose of coercing asymmetric orsymmetric mitosis) because they harbor less factors to be interferedwith.

FIGS. 5A and 5B show segregation of certain cellular factors duringasymmetric versus symmetric mitosis. Large circles represent individualstem cells, the medium-sized circle represents a differentiated progenycell of a stem cell, smaller circles represent cellular factors (e.g.,but not exclusive to, numb or notch proteins) that are apportioned todaughter cells during mitosis.

FIG. 5A depicts asymmetric mitosis, whereby certain intracellularfactors are unequally apportioned to the differentiated daughter cell.

FIG. 5B depicts asymmetric mitosis, whereby the same intracellularfactors are equally apportioned to both daughter cells.

FIGS. 6A and 6B depict sparing normal stem cells of toxicity derivedfrom is OSES-based induction of symmetric differentiation/apoptosis of acancer stem line. Large circles represent individual stem cells, themedium-sized circle represents a differentiated progeny cell of a stemcell, smaller circles represent cellular factors (e.g., but notexclusive to, numb or notch proteins) that are apportioned to daughtercells during mitosis. PTF=pre-treatment factor; AMIF=asymmetric mitosisinhibiting factor.

FIG. 6A depicts pretreatment of normal stem cells and a cancer stem linewith PTF (designed to bind a cellular factor apportioned to daughtercells) will result in loss of the PTF by normal stem cells afterasymmetric mitosis, and maintenance of the PTF by a cancer stem lineafter symmetric mitosis.

FIG. 6B depicts subsequent treatment of normal stem cells and a cancerstem line with AMIF will inhibit asymmetric mitosis only in a cancerstem line and not in normal stem cells (since, by design, the AMIFrequires the presence of the PTF for function). In this manner,induction of differentiation or starvation (not shown in figure) willresult in symmetric differentiation and/or apoptosis only of the cancerstem line while sparing normal stem cells.

DETAILED DESCRIPTION OF THE INVENTION Stem Cells and Cancer

As noted supra, the present invention relates to the realization thatthe origin of cancer involves the birth and subsequent persistence of apopulation of slow-growing relatively mutationally-spared cancer cells,i.e., the cancer stem line. Such slow-growing relativelymutationally-spared cancer cells are directly derived from normal stemcells via a one-step switch in mitotic mode, to be described in moredetail. It is this origin which explains the growth pattern of a cancerstem line (i.e., like normal stein cells from which it is derived, acancer stem line is slow-growing but rears a large transit population ofhighly proliferative cells. It is also these properties of a cancer stemline, inherited directly from normal stem cells, that will determine thenovel OSES-based methods to contain it and by doing so to treat humancancer.

Normal adult stem cells are “embryonic-like” remnants of earlydevelopment which function in adult tissues as founder cells responsiblefor cell lineage development/-tissue renewal (Potten et al, “AComparison of Cell Replication in Bone Marrow, Testis, and Three Regionsof Surface Epithelium”, Biochim. Biophys. Acta, 560:281-299 (1979);Wolpert, “Stem cells: a problem in asymmetry”, J. Cell Sci. Suppl.,10:1-9 (1988)). These poorly-differentiated immortal cell types residein well-defined environmental niches localized to the basal layer ofrenewing tissue types including semi-niferous tubules (in the case ofprimordial germ cells) as well as epidermis, intestinal crypts, andmammary terminal ducts among others. It is within these sequesteredregions that stem cells ensure proper tissue renewal, as describedindependently by Potten and Wolpert, by maintaining a constant foundercell population while concomitantly replacing aged cells and in doing socreating a local microenvironment wherein maturing/differentiatingprogeny cells migrate away from a fixed stem cell position (Potten etal, “A Comparison of Cell Replication in Bone Marrow, Testis, and ThreeRegions of Surface Epithelium”, Biochim. Biophys. Acta, 560:281-299(1979); Wolpert, “Stem cells: a problem in asymmetry”, J. Cell Sci.Suppl., 10:1-9 (1988)).

In some ways this well-defined stem cell microenvironment resembles thatof developing embryonic tissues. For example, as adult tissue renewal isfueled ultimately by the production of differentiated cells by anenvironmentally-sequestered stem cell, a developing embryonic germ layersimilarly forms from cellular descendants of a region of spatiallyconfined progenitor cells, in this case at the posterior end of amammalian embryo. In addition, the local architecture of adeveloping/renewing adult cell lineage, characterized by progressivemigration of maturing stem cell progeny from a fixed stem cell position,resembles that of developing embryonic trophectoderm and endodermwherein directed movement of cell progeny and consequent alterations intheir relations with neighboring tissues results in a restriction ofpotency (Spemann (ed.), “Embryonic Development and Induction”, New York:Hafner, Inc. (1962); Wolpert, “Positional information and patternformation”, Phil. Trans. Roy. Soc. Lond., Ser B., 295:441-450 (1981);Gurdon, J. B. Embryonic induction—molecular prospects, Development,99:285-306 (1987); Gardner et al, “Multilineage ‘stem’ cells in themammalian embryo”, J. Cell Sci. Suppl., 10:11-27 (1988)).

Based on these observations, it is also theorized that disruption of thewell-defined microenvironment of an adult stem cell may have similarconsequences to altering the surroundings of an embryonic cell. As willbe discussed (and as mentioned earlier), there are several classicexperiments which demonstrate that alterations to the surroundings of adeveloping embryonic tissue can result in aberrant development and“neoplasia” (Rubin, “Cancer as a Dynamic Developmental Disorder”, CancerRes., 45:2935-2942 (1985); Pierce et al (eds.), “Cancer: a problem ofdevelopment biology”, New Jersey: Prentice Hall Inc. (1978)).Accordingly, it is theorized here that the disturbance of the localenvironment of a stem cell (in manners to be discussed) could, in asimilar way to disrupting an embryonic environment, predispose potentcell types (i.e., stem cell in this case) to aberrant development and“neoplasia”. An examination of some of the recently-described molecularmechanisms by which embryonic and adult cell lineages normally developas well as some of the potential mechanisms by which they can go awrysupports this theory.

During embryogenesis, one role of the surrounding spatial architectureof developing tissues is to act as a dynamic scaffold that directsdifferentiation, morphogenesis, and fulfillment of a properdevelopmental plan (Spemann (ed.), “Embryonic Development andInduction”, New York: Hafner, Inc. (1962); Wolpert, L., Positionalinformation and pattern formation, Phil. Trans. Roy. Soc. Lond., Ser B.,295:441-450 (1981); Gurdon, “Embryonic induction—molecular prospects”,Development, 99:285-306 (1987); Gardner et al, “Multilineage ‘stem’cells in the mammalian embryo”, J. Cell Sci. Suppl., 10:11-27 (1988)).Mechanistically, it had been previously suggested by Holtzer thatdeveloping embryonic cells might limit their potency and differentiate,when surrounded by a proper inducing environment (i.e., scaffolding),via a switch from symmetric to asymmetric mitotic division leading toformation of differentiated cell types (Stamatoyannopoulos et al (eds.),“Globin Gene Expression and Hematopoietic Differentiation”, pp. 213-227.New York: A. R. Liss, Inc. (1983)).

There is now indeed strong evidence that certain keydevelopmentally-regulated cell fate decisions during embryogenesis aremediated specifically by a timely switch from symmetric to asymmetricmitotic division. This process has been likened to a highly conservedone-step epigenetic mitotic switch, as described by Herskowitz, thatregulates vegetative growth in lower eukaryotes (Horvitz et al,“Mechanisms of Asymmetric Cell Division: Two Bs or Not Two Bs, That isthe Question”, Cell, 68: 237-255 (1992)). Mechanistically, a variety ofintracellular proteins (e.g., NOTCH and m-NUMB) have been found to beunequally segregated to daughter cells at asymmetric mitosis duringmammalian development. These findings coupled with the demonstrationthat equal segregation or, alternatively, loss of expression of theseand other related factors can lead to a switch in mitotic mode fromasymmetric to symmetric division strongly indicates a causal role (bypreferentially segregated intracellular factors) in determining the fateof their host cell. Moreover, the proper apportioning of suchintracellular factors has been shown to be affected by certain extrinsiclocally-acting factors and their downstream is signaling pathwaysthereby providing molecular support for past proposals thatmorphogenesis is dependent on a proper surrounding environment (i.e.,scaffold) wherein intercommunication between developing embryonic celltypes occurs, e.g., presumably by triggering a switch from symmetric toasymmetric embryonic cell mitosis (Lin et al, “Neuroblasts: a model forthe asymmetric division of stem cells”, Trends Genet., 13: 33-39 (1997);Wolpert, “Positional information and pattern formation”, Phil. Trans.Roy. Soc. Land, Ser B., 295: 441-450 (1981)). It would follow from thisidea that disruption of the surrounding environment of developingembryonic cells (e.g., via ectopic transplantation of embryonic cellsout of their native environment) would disturb developmentally-regulatedlocal induction of asymmetric embryonic cell mitosis which in turn couldallow such developing cells to continue to proliferate via symmetricmitosis (i.e. exponentially)—an aberrant process which would resemble“neoplasia”.

As previously mentioned, when embryonic ectoderm is ectopicallytransplanted to adult tissues it can form a neoplasm (i.e.,teratocarcinoma) which then regresses if replaced back into theembryonic milieu of a developing blastocyst (Pierce et al (eds.),“Cancer: a problem of developmental biology”, New Jersey: Prentice HallInc. (1978)). By conventional models, one might have to invokemutation-selection to create this neoplasm followed by a enigmatic“bypassing” of such permanent genomic derangements to account for itsreversibility. However, consider an alternative novel explanationwhereby placement of ectodermal cells into a foreign tissue milieuresults in an interruption of native signals that normally induce themto undergo asymmetric mitosis thereby leading to persistence ofsymmetric mitoses, i.e., effectively a one-step epigenetic switch (OSES)from asymmetric to symmetric embryonic cell mitosis, resulting insymmetric mitoses out of proportion to asymmetric mitoses, a netproliferation of misplaced embryonic cells, and a disorderly/neoplastic(e.g., “teratocarcinomatous”) phenotype. In this manner, thereversibility of certain teratocarcinomas upon placement intoblastocysts, rather than reflecting a “bypass” of permanent mutations,could indicate that this tumor is no more than a collection of misplacedectodermal cells which maintain a capability to resume propermorphogenesis upon restoration of endogenous differentiation-inducingsignals (because they did not amass mutational changes and thus did notneed to bypass any). Assuming embryonic and adult tissue development aremechanistically related processes, as alluded to, then disruption of thelatter process (e.g., via carcinogen-induced tissue damage), likedisturbance of the former (e.g., via ectopic transplantation) might alsolead to aberrant epigenesis and neoplasia via a similar mechanism.

Like their developing embryonic counterparts, there is evidence thatadult stem cells act via a similar asymmetric mitotic process which alsorelies on a signaling pathway induced by extrinsic cues derived fromneighboring cells (and/or extracellular matrix) (Lin et al,“Neuroblasts: a model for the asymmetric division of stem cells”, TrendsGenet., 13: 33-39 (1997); Potten et al, “A Comparison of CellReplication in Bone Marrow, Testis, and Three Regions of SurfaceEpithelium”, Biochim. Biophys. Acta, 560: 281-299 (1979); Wolpert, “Stemcells: a problem in asymmetry”, J. Cell Sci. Suppl., 10: 1-9 (1988)).However, in contrast to the relatively “loose” architectural arrangementof developing embryonic tissue and seemingly stochastic manner by whichdeveloping embryonic cells asymmetrically produce less potent progenythat differentiate, the well-defined microenvironmental stem cell nichemay make stem cells subject to a relatively more continual flow ofextrinsic inducing signals thereby resulting in a relatively lessstochastic process characterized by regular asymmetric mitoses at thecompletion of each and every stem cell-cycle. (See FIG. 1A)

Thus, by this model, despite the clear architectural difference betweenembryonic and adult tissues, the processes of embryogenesis and adulttissue renewal can still be analogous and driven by similar mechanisms(i.e., symmetric and timely asymmetric mitoses). Accordingly,disturbance of an embryonic milieu (e.g., due to ectopic ectodermal celltransplantation) will result in tumor formation due to maintenance of anembryonic phenotype arising from a preponderance of symmetric embryoniccell mitoses. Similarly, disruption of a stem cell microenvironmentwould also lead to neoplastic growth arising from persistence of a stemcell phenotype due to continued symmetric stem cell mitoses. Morespecifically, disturbance to the regular flow of local inducing signalsnecessary for ensuring regular asymmetric stem cell mitoses, adisturbance arising possibly from carcinogen-induced damage of adeveloping stem cell milieu (a microenvironment which consists largelyof non-stem cells responsible for providing such inducing signals totheir stem cell neighbors) will result in disruption of this normallytightly-controlled process thereby leading effectively to a one-stepepigenetic switch (OSES) from asymmetric to symmetric stem cell mitoses.(See FIG. 1B)

It also follows from such a model that symmetrically—dividing stem cells“crowded” within a stem cell niche would be correspondingly subject tolocal inducing signals which normally only affect the fate of a singlesequestered stem cell. In this manner, symmetrically-dividing stem cellswould in effect “overload the system” (i.e., too many cells and notenough differentiation-inducing signals) thereby causing them to adopt amore stochastic mode of differentiation-induction characteristic ofdeveloping embryonic tissues. Accordingly, the timing of induction ofasymmetric stem cell mitosis within a disrupted milieu would no longerbe regular thereby potentially leading to net proliferation, unplanneddifferentiation, and a disorderly-appearing tissue mass. By this model,the phenotype of symmetrically-dividing adult stem cells, of thegermline as an example (i.e., primordial germ cells), would approximatethe teratocarcinomatous appearance proposed for symmetrically-dividingectodermal cells when ectopically transplanted thereby obviating theneed to invoke a multi-step evolutionary process in the birth of thistumor from primordial germ cells. In this manner, the unstructuredhistopathologic phenotype characteristic of early solid tumors couldrepresent pre-existing cancer cells and theirirregularly-differentiating progeny rather than a collection of“pre-cancerous” intermediates. Accordingly, symmetrically-dividing adultstem cells are synonymous with cancer cells.

Interestingly, there is evidence that stem cells are the cell type oforigin for a variety of hematologic as well as solid malignancies whichbegs the question as to how many more alterations would be required bystem cells, which are already poorly-differentiated and “immortal”, toassume a neoplastic phenotype? It had been previously described byPierce that certain cancer cell types were no less differentiated at thehistopathological level than stem cells from their correspondinglineages of origin. In addition, the renewal rates of a variety ofdeveloping tissues known to harbor stem cells (e.g., bone marrow,gastrointestinal tract, and testis) have been reported to be comparableto the exponential growth rates of corresponding cancer cell typesderived from these tissues (Sell et al, “Maturation Arrest of Stem CellDifferentiation as a Common Pathway for the Cellular Origin ofTeratocarcinomas and Epithelial Cancers”, Lab. Invest., 70:6-22 (1994));Pierce et al (eds.), “Cancer: a problem of development biology”, NewJersey: Prentice Hall Inc. (1978)). Based on these mentioned findings itappears that the major difference between a stem cell and a cancer cellarising from the same tissue of origin is the mode by which these twopoorly-differentiated immortal cell types divide (i.e., asymmetricmitosis leading to arithmetic cellular growth vs. symmetric mitosisleading to exponential cellular growth, respectively), anepigenetically-derived trait.

Since there is evidence that the developmental decision to switch fromsymmetric to an asymmetric mitotic mode can occur in one-step via anepigenetically-controlled mechanism (Horvitz and Herskowitz, “Mechanismsof Asymmetric Cell Division: Two Bs or Not Two Bs, That is theQuestion”, Cell, 68:237-255 (1992)), multiple mutational derangementsare likely unnecessary for an aberrant switch from the latter to theformer to occur in developing adult tissues (i.e., stem cells).Comparison of the respective genomes and expression profiles of stemcells and cancer cells using conventional molecular biologicaltechniques (e.g., subtraction hybridization) will allow one to moreaccurately substantiate the noted gross (morphological and mathematical)similarities between these cell types.

Support for the proposed OSES model can be sought by experimentallyinducing stem cells to divide symmetrically and observing whether theresultant phenotype resembles neoplasia. Since the phenotype of a stemcell is usually described in the context of arithmetic cell divisionleading to orderly tissue renewal without a net increase in cell number(i.e., asymmetric mitosis) it has not yet been shown what consequenceswould arise if the phenotype of a stem cell were to persist in both ofits daughter cells (i.e., symmetric mitosis). If local extrinsic signalsare responsible for ensuring regular asymmetric stem cell mitoses thenone might have to experimentally disperse stem cells from theirsequestered niches in order to examine a true stem cell phenotypewithout environmental influence. Namely, dissection of certain adultstem cells out of their well-defined microenvironments, according to theOSES model, should result in loss of local inductive signals requiredfor asymmetric stem cell mitosis (as with transplanted embryonic cells)thereby leading to a switch to symmetric stem cell mitoses and tumorformation. In addition, experimental reimplantation of such a lesionback into its native stem cell microenvironment should effectcarcinogenic reversion and resumption of normal lineage development—aphenomenon previously demonstrated for teratocarcinomas (Pierce et al(eds.), “Cancer: a problem of development biology”, New Jersey: PrenticeHall Inc. (1978)). Stem cells of the hematologic lineage have beensuccessfully purified using various monoclonal antibody techniquesthereby making the proposed experiments feasible at least for thehematopoietic system (Uchida et al “Heterogeneity of hematopoietic stemcells”, Curr. Opin. Immunol., 5: 177-184 (1993). There has also beensome success in transplanting stem cells of non-hematologic tissues(e.g., intestinal epithelium) but purification of these cells awaitsfurther technical improvements (Gordon, “Differentiation andself-renewal in the mouse gastrointestinal epithelium”, Curr. Opin. CellBio., 6: 795-803 (1994)). Antigens and other specific gene productsdifferentially expressed by stem cells and their differentiated progenyin a variety of different cell types are being identified at anever-increasing pace (e.g., integrins in developing skin) and could actas potential molecular targets for the purpose of separating stem fromnon-stem cells in preparation for transplantation to test the OSES model(Sigal, “The liver as a stem cell and lineage system”, Am. J. Physiol.,263: G139-G148 (1992); Jones et al, “Stem Cell Patterning and Fate inHuman Epidermis”, Cell, 80: 83-93 (1995)).

Closer examination of some previous reports of tumor formation thatfollows ectopic transplantation of certain adult tissues (e.g.,pituitary and testis) to foreign tissue locales should confirm (or deny)through utilization of more current techniques that the neoplasms thatarise from such transplants are derived specifically from the stem cellsof these tissues as would be predicted by the OSES model (Rubin, “Canceras a Dynamic Developmental Disorder”, Cancer Res., 45:2935-2942 (1985);Farber and Rubin, “Cellular Adaptation in the Origin and Development ofCancer”, Cancer Res., 51:2751-2761 (1991)). Moreover, experimentalretransplantation of these tumors back within the confines of the stemcell microenvironments of their respective tissues of origin shouldconfirm whether recreation of normal cell lineage development, aspredicted by the OSES model, can occur.

In summary, by the OSES model, as discussed, cancer “initiation” iscaused by (e.g., carcinogen-induced) damage to a developing stem cellmilieu, and more specifically from damage to those (non-stem) cells thatnormally induce their stem cell neighbors to undergo regular asymmetricmitoses. Since a considerable proportion of the non-stem cells whichsurround a stem cell are a stem cell's own progeny, might damagespecifically to this particular cell population lead indirectly to astem cell-derived cancer? In this manner, perpetuation of a mutantdifferentiation—related gene by a stem cell to its non-stem cell progeny(but not expressed in a stem cell which does not itself differentiate)could lead to aberrant cell differentiation (due to expression of amutant differentiation-related genes in differentiating cells) and andisrupted stem cell microenvironment (due to aberrantly differentiatingcells within the milieu). This scenario in turn like that of embryoniccells placed in a foreign milieu, will permit developing (stem) cells tobecome neoplastic by predisposing them to symmetric mitoses. It followsfrom this idea that germline inheritance of certain mutantdifferentiation-related genes (e.g., TP53, RB-1, BRCA-1) might lead to anovel non-neo-Darwinian manner in which to endow a cancer predispositionwhereby such genes adversely affect the differentiation capabilities ofnon-stem cells thereby predisposing neighboring stem cells to symmetricmitoses. Evidence that an inherited cancer predisposition can beaffected by mutagenic effects on (non-neoplastic) cells which neighbor aneoplasm is provided by Varmus' group wherein some mammary tumorsderived from germline TP53/Wnt-1 altered mice may be more aggressivewhen surrounded by p53−/− cells than by “less-mutated” p53+/−cells(Donehower et al, “Deficiency of p53 accelerates mammary tumorigenesisin Wnt-1 transgenic mice and promotes chromosomal instability”, GenesDev., 9: 882-895 (1995)). However, are these proposals by the OSES modelalso consistent with well-corroborated data correlating mutagenesis withthe latter “progression” phase of cancer development?

Along similar lines, if symmetrically-dividing stem cells (i.e., cancercells) are susceptible to local differentiation-inducing signals (whichwould induce tumor regression) then how could a mass of such cellscontinue to grow in size and eventually invade, i.e., “progress” ratherthan regress?

Tumor Progression

It is a well-documented phenomenon that most tumor types evolve at thetissue level from a “benign-appearing” to an increasingly“cancerous-appearing” lesion (Furth, “Conditioned and autonomousneoplasm: a review”, Cancer Res., 13: 477-492 (1953); Foulds, “Thenatural history of cancer”, J. Chronic Dis., 8: 2-37 (1958)). Thismetamorphosis could in theory result from either the stepwise evolutionof increasingly cancerous cellular intermediates (i.e. conventionalmodel) or alternatively from the gradual emergence in numbers of asubpopulation of cancer cells created via a one-step non-evolutionaryprocess (i.e. the OSES model). The correlation between mutationalaccumulation and tumor “progression” at the tissue level has been citedin support of the former idea (Weinberg, “Oncogenes, Anti-oncogenes, andthe Molecular Basis of Multistep Carcinogenesis”, Cancer Res., 49:3713-3721 (1989); Nowell, “The Clonal Evolution of Tumor CellPopulations.”, Science (Washington D.C.), 194: 23-2.8 (1976)).Additional support for neo-Darwinian cellular evolution has arisen fromrecent high resolution molecular analyses of human tumor biopsyspecimens revealing that adjoining “pre-cancerous” and “cancerous” tumorregions can share certain rare mutational alterations—findings cited asevidence for a direct lineage from a “benign” cell to a “cancerous” one.Moreover, demonstration of the presence of certain unique geneticalterations solely within “cancerous” portions of a tumor has beeninterpreted as further evidence of a causal role for mutation ineffecting the cellular transition from “benign” to “malignant” (Fearonand Vogelstein, “A Genetic Model for Colorectal Tumorigenesis”, Cell,61:749-767 (1990); Sidransky et al, “Clonal expansion of p53 mutantcells is associated with brain tumor progression”, Nature (Lond.),355:846-847 (1992)).

However, these mentioned observations cited in support of neo-Darwiniancellular evolution are also consistent with the OSES model. Namely,while the presence of a rare mutation within both a “benign-appearing”and an adjoining “cancerous-appearing” portion of a tumor may beinterpreted as evidence for conventional neo-Darwinian evolution at thecellular level, this is not the only explanation for such a finding.Rather, the sharing of a rare mutation by adjacent tumor regions maymerely reflect a common ancestry for cells within such neighboring siteswithout definitively revealing which cells arose from which.Accordingly, a reverse temporal scenario to that of conventionalmutation selection but consistent with the OSES model is possiblewhereby a direct lineage between cells within adjacent “benign” and“cancerous” tumor regions is maintained but via reversion of “cancerous”cells into “benign” ones (e.g., via cancer cell differentiation), ratherthan via progression of the latter to the former. In this way, mutationspresent in adjoining “benign” and “cancerous” tumor regions would beconsidered essentially “neutral”, i.e., not adversely affecting theability of cancer cells to is differentiate. In a related manner,detection of unique mutations solely within a “cancerous” region of atumor, although cited in support of conventional mutation-selection, isalso consistent with the OSES model which explains these geneticalterations as acquired features of pre-existing cancer cells ratherthan as effectors of “progression” toward malignancy. Thus, suchmutations present solely within a “cancerous” portion of a tumor wouldrepresent selectively advantageous genetic alterations that inhibitcancer cell differentiation/regression into “benign” cells. In thismanner, such advantageous mutations might be expected to hasten theemergence of a clone of pre-existing cancer cells from amongst itsslowergrowing (i.e. differentiating/regressing) non-mutant neighbors. Sothe presence of clonal mutant outgrowths in tumors, a finding oftencited in support of conventional neo-Darwinian models, is alsoconsistent with the OSES model. In other words, these mentionedtumor-related data alone are insufficient to categorically determine thelineage history of related tumor cell populations and thus do not ruleout the idea of differentiation from “cancerous” to “benign” (i.e., theOSES model). Accordingly, higher resolution lineage analyses, comparingrespective tumor regions using multiple independent cellular markers,may be required to better discern between these two divergent accounts.

By the OSES model then the phenomenon of tumor “progression” wouldcorrespond to a gross histopathologic change due to the gradualemergence of preexisting cancer cells rather than the stepwise selectionof individual “pre-cancerous” intermediates. The preferential outgrowthof a clone(s) of cancer cells from amongst its slower-growing (i.e.differentiating/regressing) neighbors could then be attributed to theadvantageous acquisition of mutations which inhibit that mutant clone'sability to differentiate. More specifically, by the OSES modelperipherally-located cancer cells in a tumor mass may be the mostsusceptible population to selective pressure fordifferentiation-impairing mutations thereby effectively “shielding” themore centrally-located cancer cells of such pressures (and thus ofsignificant mutational accumulation). Accordingly, expansion ofperipherally-located mutants which are differentiation-defective (andwhich may or may not have a limited proliferative capability) mightfurther shield a more centrally-located subpopulation'thereby allowingit to expand and act as an “immortal” founder line (i.e. cancer stemline) that is relatively mutationally-spared. This idea is consistentwith reports that certain human tumors (e.g., breast carcinomas) have ahigher histopathological grade in their more central regions as well aswith findings that some highly aneuploid tumor types have a chromosomaldistribution pattern best accounted for by the presence of a stem linewhich is neareuploid (Lennington et al, “Ductal carcinoma in situ of thebreast. Heterogeneity of individual lesions”, Cancer, 73:118-124 (1994);Makino, “Further Evidence Favoring The Concept of the Stem Cell InAscites Tumors Of Rats”, Ann. N.Y. Acad. Sci., 63:818-830 (1956);Shapiro et al, “Isolation, Karyotype, and Clonal Growth of HeterogeneousSubpopulations of Human Malignant Gliomas”, Cancer Res., 41:2349-2357(1981)). If some mutations could affect tumor growth behavior subsequentto the birth of a cancer cell (e.g., by inhibiting cancer celldifferentiation) then another potential non-neo-Darwinian explanationfor an inherited cancer predisposition (in addition to predisposing tothe early “initiation” stage of cancer by causing non-stem cells toaberrantly differentiate) might also derive from germline inheritance ofa mutant gene that accelerates the latter “progression” stage of cancerby thwarting differentiation/reversion of cells which are a cancerous.

It should be noted that it had been previously described by Pierce thatcertain malignant cell types including neuroblastoma, leukemia,rhabdomyosarcoma, and mammary adenocarcinoma are capable of“differentiating” into benign tissues. Namely, classic experimentsinvolving thymidine labeling of rat squamous cell carcinomas and singlecell cloning of murine teratocarcinomas revealed that thepoorly-differentiated cells within these particular tumor types hadgiven rise to well-differentiated tumor cells but not vice versa (i.e.,not via dedifferentiation). It was further shown in these experimentsthat the poorly-differentiated cells within these neoplasms were thecell types responsible for tumor growth and invasion while their welldifferentiated progeny had essentially lost malignant growth potentialand were thus deemed to be “benign” (Pierce et al (eds.), “Cancer: aproblem of development biology”, New Jersey: Prentice Hall Inc. (1978)).A question not adequately addressed at the time but which may be able tobe evaluated by current molecular techniques is whether the “benign”cells within these particular experimental rodent tumors are analogousto those “benign” cells normally classified as “pre-cancerous” in humantumors. In other words, as hypothesized here, cells within a“benign-appearing” region of human tumors may actually constitutedifferentiated progeny of pre-existing cancer cells rather than“pre-cancerous” intermediates. Re-exploration of these mentioned earlyexperiments with current molecular biological techniques shouldelucidate whether rodent tumors previously shown to harbor evidence ofcancer cell differentiation also possess a mutational distributionsimilar to that described for human neoplasms—a distribution that hasbeen attributed to conventional mutation-selection. Detection of asimilar mutational distribution should provide further evidence that theOSES model (which invokes cancer cell differentiation) provides anequivalent explanation as neo-Darwinism for the presence of“benign-appearing” cells within human cancers—and a better explanationthan conventional models for cases of epigenesis in carcinogenesis (asargued previously).

It is also noted that there are cases in which cancer-related mutationsdo not clearly accumulate with tumor “progression”. Such data have notbeen readily accounted for by conventional models, but when analyzedmore closely, are indeed consistent with the predictions of the OSESmodel.

Mutational Accumulation Without “Progression”

As mentioned, the well-documented correlation between mutationalaccumulation and cancer development might in theory be accounted for bymore than one model (i.e. neo-Darwinism or OSES). However, there alsoexists data of a different type wherein mutational accumulation does notalways proceed in sync with the early stages (i.e. “initiation”) orlatter stages (i.e. “progression”) of cancer development. Such findings,as will be described in part are difficult to reconcile by a classicalneo-Darwinian paradigm which, by definition, invokes selection as theprimary explanation for the presence, in large numbers of cells, ofmutations that would otherwise be rarely found.

For example, there are some newly described enigmatic cancer-relatedphenomena wherein the presence of detectable mutations at an early stageof cancer development does not always signify “selection”. Namely, avariety of genomic alterations have been detected not only in cancercells but surprisingly also in synchronous histologically normal cellsof the same tissue type (e.g., loss of heterozygosity (LOH) of WT-1 orhypermethylation of H19 in kidney, LOH of breast cancer-related loci inmammary gland, and mutator phenotype (MT) defects in colon) (Parsons etal “Mismatch Repair Deficiency in Phenotypically Normal Human Cells”,Science (Washington D.C.), 268: 738-740 (1995); Chao et al, “Geneticmosaicism in normal tissues of Wilms' tumor patients”, Nature Genet., 3:127-131 (1993); Moulton et al, “Epigenetic lesions at the H19 locus inWilms' tumor patients”, Nature Genet., 7: 440-447 (1994). Deng et al,“Loss of Heterozygosity in Normal Tissue Adjacent to Breast Carcinomas”,Science (Washington D.C.), 274: 2057-2059 (1996)). By conventionalmodels, rare mutations become detectable predominantly by selectionalone. Accordingly, the data mentioned above provide somewhat of aparadox for conventional thinking which considers detectable mutationsby definition to be selectively advantageous, and that thosenon-neoplastic cells harboring such genetic changes should thereforedisplay some evidence of “overgrowth” histopathology—a prediction whichis not supported by these mentioned studies. These findings are,however, consistent with the OSES model which predicts that mutationsact in “initiation” not by accumulating in and promoting growth offuture cancer cells (i.e. stem cells) but rather by causing geneticchanges in neighboring non-stem cells which impair their differentiationcapability—a process that in turn leads to an altered tissue milieupredisposing to symmetric stem cell mitoses. In this manner, thereshould be a lag period characterized by detectable mutagenesis (followedby aberrant differentiation) before any histopathologic evidence ofproliferative activity is evident. This scenario predicted by the OSESmodel, unlike the one predicted by conventional models, is consistentwith the mentioned studies.

In a related manner, there are several other reports, as alluded to,wherein mutations also do not entirely correlate with the latter stagescancer evolution. For example, a subset of human colorectal tumorspossess functional alterations at some cancer-related loci (e.g.,c-K-RAS or TP53) in “benign” but surprisingly not in adjacent“cancerous” regions (Shibata et al, “Genetic Heterogeneity of thec-K-ras Locus in Colorectal Adenomas but not in Adenocarcinomas”, J.Natl. Cancer Inst., 85: 1058-1062 (1993); Ohue et al, “A FrequentAlteration of p53 Gene in Carcinoma in Adenoma of Colon”, Cancer Res.,54: 4798-4804 (1994); Kaklamanis et al, “p53 Expression in ColorectalAdenomas”, Am. J. Path. 142: 87-93 (1993)). Similarly, LOH at variousloci have been detected in pre-invasive but not adjoining invasiveregions of a subset of human breast carcinomas (O'Connell et al,“Molecular genetic studies of early breast cancer evolution”, BreastCancer Res. Treat., 32: 5-12 (1994)). In a related manner, a subset ofprimary gastric tumors with mutant TP53 display only wildtype TP53 atmetastatic foci (Strickler et al, “p53 Mutations and MicrosatelliteInstability in Sporadic Gastric Cancer: When. Guardians Fail”, CancerRes., 54: 4750-4755 (1994)). By the OSES model “cancerous” cells could,upon induction by local differentiation-inducing signals (as mentionedin the previous section) pass on a “neutral” mutation to a largepopulation of its “benign” progeny. This idea would explain theseemingly enigmatic presence of certain mutations in a “less-cancerous”region but not in a correspondingly “more-cancerous” region. Byconventional thinking however, one may have to infer from these datathat either cells within “more-cancerous” regions had not arisen fromones within “less-cancerous” regions, or that neoplastic cells wild-typeat a given locus had somehow out-competed their corresponding mutant. Byeither of these explanations, the classic clonal selection hypothesis byNowell is violated (Nowell, “The Clonal Evolution of Tumor CellPopulations”, Science (Washington D.C.), 194: 23-28 (1976)).Accordingly, in an effort to maintain a neo-Darwinian model, one mayhave to speculate that a “surreptitious” mutator phenotype (MP)-likemechanism may have acted in a (seemingly wild-type) clone to enable itsselection over detectably-mutant competitors—an idea which awaitsexperimental support. However, while combining conventional geneticalterations (e.g., in c-K-RAS or TP53) with an MP-hike mechanism mightappear to provide an adequate neo-Darwinian explanation for these data,this idea clashes with observations that these distinct mutationalmechanisms usually do not coexist within the same tumor (Perucho,“Microsatellite instability: The mutator that mutates the othermutator”, Nature Med., 2: 630-631 (1996). Accordingly, conventionalneo-Darwinian models do not adequately account for these mentionedcases—cases which are more completely explained by the OSES model.

A discussion of some of the testable differences between the OSES andconventional models is set forth below.

Testable Differences Between the OSES and Conventional Cancer Models

There are a number of testable differences between the OSES model andconventional cancer models. Some of these differences are discussedbelow.

The expression profile of a stem cell is inherited en bloc by a cancercell. Based on the proposal that a one-step epigenetic switch (OSES)from asymmetric to symmetric stem cell mitosis is responsible for thebirth of a cancer cell, it follows from such a model that cancer cellswill inherit an expression profile largely en bloc from stem cells. Inthis manner, the expression profile of a cancer cell will be generallythe same as that for a stem cell, i.e., no major epigenetic ormutational alterations will be incurred, with the caveat that a stemcell by virtue of its environmental sequestration passes its phenotypicstate on to only one of its daughter cells via asymmetric mitosiswhereas a cancer cell (i.e., symmetrically-dividing stem cell) passesits inherited stem cell phenotypic state on to both daughter cells viasymmetric mitosis unless extrinsically induced to transiently divideasymmetrically by local signals. In contradistinction to previouslydescribed epigenetic models as well as current neo-Darwinian models forcancer which each invoke a stepwise accumulation of heritable cellularalterations in the evolution of a cancer cell, the OSES model depictsthe cancer cell phenotype not as a collection of mutational and/orepigenetic traits acquired in a stochastic piecemeal fashion but ratheras a state inherited directly from a stem cell precursor in one stepwithout the need for further changes (Holliday, “A New Theory ofCarcinogenesis”, Br. J. Cancer, 40:513-522 (1979); Rubin, “Cancer as aDynamic Developmental Disorder”, Cancer Res., 45:2935-2942 (1985);Vogelstein et al, “The multistep nature of cancer”, Trends Genet.,9:138-141 (1993)).

Differentiation-Related Genes are Inherited by Cancer Cells from StemCells Largely in an Epigenetically Down-Regulated State.

Genes which do not function in maintenance of the stem cell phenotype(e.g., genes involved in enactment of differentiation/asymmetricmitosis) are likely to be normally down-regulated in stem cells duringmost of their cell cycle until G1-S at which time they become upregulated in preparation for asymmetric production of differentiatedprogeny (Villarreal, “Relationship of Eukaryotic DNA Replication toCommitted Gene Expression: General Theory for Gene Control”, Microbiol.Rev., 512-542 (1991)). Therefore, according to the OSES modeldifferentiation-related gene complexes will be inherited by cancer cellsfrom stem cells en bloc in an epigenetically down-regulated state. Inthis manner, stochastic acquisition by cancer cells of mutations todifferentiation-related loci would not initially be expected to endowthem with any particular selective advantage (or disadvantage) sincethese gene complexes should be down-regulated/unexpressed anyway.However, by the OSES model, if a cancer cell was subsequentlyextrinsically induced by local signals to asymmetrically producedifferentiated progeny then its differentiation-related genes would bepredicted to become transiently up-regulated. If these genes had beensignificantly mutationally damaged while unexpressed then subsequentup-regulation of such mutant genes upon enactment of a properdifferentiation program would lead to a defective differentiationcapability thereby endowing such mutant cancer cells with a selectivegrowth advantage over their differentiating neighbors.

There is evidence in support of these proposals, i.e. that a cancer cellexpression profile is inherited en bloc from a stem cell and thatdifferentiation-related pathways in particular are inherited by a cancercell from a stem cell in a down-regulated-state. For example, the are anumber of reports indicating that certain gene complexes, namely thoseinvolved in differentiation, may be largely down-regulated in stem cellsfor the majority of their cell cycle (Wolpert, L., Stem cells: a problemin asymmetry, J. Cell Sci. Suppl., 10:1-9 (1988); Villarreal,“Relationship of Eukaryotic DNA Replication to Committed GeneExpression: General Theory for Gene Control”, Microbiol. Rev., 512-542(1991)). It follows from the OSES model that a cancer cell shouldmaintain the expression profile of its precursor stem cell and thereforebe expected to harbor such gene complexes in their native down-regulatedstate. Demonstration that certain differentiation-related genes areinitially epigenetically down-regulated in both stem cells andcancer-cells, then up-regulated during (stem cell or cancer cell)differentiation, and subsequently mutated following the birth of acancer cell rather than before is consistent with and stronglysupportive of a temporal scenario predicted by an OSES model forcarcinogenesis.

The OSES cancer model can be further substantiated by studyingdifferentiation-related gene complexes, e.g., TS suppressor genecomplexes. There is evidence that several tumor suppressor genes (TS's),i.e., genes notoriously found mutationally inactivated or deleted incancer cells, may comprise one such group of genes that normallyfunctions in enactment of differentiation/cell lineage development(Schmid et al, “Expression of p53 during mouse embryogenesis”,Development, 113:857-865 (1991); Kent et al, “The evolution of WT1sequence and expression pattern in the vertebrates”, Oncogene,11:1781-1792 (1995); Cordon-Cardo et al, “Expression of theretinoblastoma protein is regulated in normal human tissues”, Am. J.Path., 114:500-510 (1994); Marquis et al, “The developmental pattern ofBrca1 expression implies a role in differentiation of the breast andother tissues”, Nat. Genet., 11:17-26 (1995)).

Such investigation would include, e.g., experiments studying whetherTS's are largely down-regulated in stem cells (during the majority oftheir cell cycle); if TS's are initially epigenetically down-regulatedin cancer cells (i.e., reflecting a state inherited directly from stemcells); and whether mutational inactivation of TS's in cancer cellsoccurs subsequent to an initial epigenetically down-regulated state.While the results of any one of these experiments alone might not besufficient to differentiate between a neo-Darwinian and an OSES model,demonstration of all three conditions in a given tumor would providestrong evidence in support of the validity of an OSES model.

Findings which would Support Novel Predictions of OSES Model.

1. Are TS's Largely Down-Regulated in Stem Cells?

There is evidence, arising largely from a combination of experimentalanalyses sought to determine both endogenous TS expression as well asthe consequences of loss of TS function, that several TS's can play keyroles in directing proper development of embryonic as well as adulttissues. For example, TP53 has been shown to function in normalhematopoiesis, WT-1 in early mesodermal differentiation and kidneydevelopment, RB-1 in developing CNS and hematopoietic systems, andBRCA-1 during mammary gland morphogenesis. Moreover, expression leveldifferences in TP53, WT-1, and RB-1 between proliferating precursor cellcompartments and their early maturing progeny have led some to proposethat timely expression level changes of certain TS's in potent cells maybe a prerequisite for the developmentally-controlled switch fromproliferation to differentiation (Schmid et al, “Expression of p53during mouse embryogenesis”, Development, 113:857-865 (1991); Kent etal, “The evolution of WT1 sequence and expression pattern in thevertebrates”, Oncogene, 11:1781-1792 (1995); Cordon-Cardo et al,“Expression of the retinoblastoma protein is regulated in normal humantissues”, Am. J. Path., 114:500-510 (1994); Marquis et al “Thedevelopmental pattern of Brca1 expression implies a role indifferentiation of the breast and other tissues”, Nat. Genet., 11:17-26(1995)). It would follow from such a model that if upregulation ofcertain TS's and/or their pathways led to enactment of a differentiationprogram then a native down-regulated state for TS's and/or theirpathways would be expected to exist in non-differentiating proliferatingcells (i.e., embryonic precursor cells or adult stem cells). That TS'scan indeed provoke cell differentiation is an idea supported by certainin vitro studies illustrating that up-regulation of TS's (e.g., TP53)induces (epithelial and hematopoietic) differentiation while expressionof mutant TS's (e.g., TP53) can lead to aberrant (thyroid cell)differentiation (Soddu et al, “Wild Type p53 Gene Expression InducesGranulocytic Differentiation of HL-60 Cells”, Blood, 83:2230-2237(1994); Battista et al, “A mutated p53 gene alters thyroid celldifferentiation”, Oncogene, 11:2029-2037 (1995); Sherley et al,“Expression of the wild type p53 anti-oncogene induces guaninenucleotide-dependent stem cell division kinetics”, Proc. Natl. Acad.Sci. USA, 92:136-140 (1995)).

While the mechanism by which either transient or permanent expressionlevel changes of certain TS's and/or their pathways can effect celldifferentiation has yet to be determined, there is some preliminaryexperimental evidence that this process can result from TS-induction ofstem cells to produce progeny destined to differentiate via anasymmetric mitotic mechanism. Namely, Sherley et al have recently shownthat up-regulation of p53 causes immortalized murine epithelial cells toswitch from an exponential to a linear growth pattern. Demonstrationthat quiescent but viable cell progeny are produced following thisswitch indicates that adoption of division kinetics characteristic ofrenewing stem cells, rather than that of cell senescence, might be themost accurate explanation for these findings. Moreover, division historyanalysis of single p53-induced cells plated at low density demonstratesindeed that a non-random mitotic process (i.e., maintenance of oneimmortal daughter cell) characteristic of asymmetric stem cell mitosiswas responsible for the p53-induced change, in cellular growth kinetics(Sherley et al, “Expression of the wild type p53 antioncogene inducesguanine nucleotide-dependent stem cell division kinetics”, Proc. Natl.Acad. Sci. USA, 92:13&140 (1995)).

Related experiments using inducible constructs of additional TS's shoulddetermine whether or not enactment of asymmetric mitosis' is a commonmechanism, of action for these genes. In addition, temporal and spatiallocalization of TS gene products in renewing tissues may ultimatelydetermine whether these genes function specifically as inducers ofasymmetric stem cell mitosis in vivo as predicted by the OSES model. Bysuch a model, TS expression level changes would be expected to bedetectable at the interface between renewing stem cells and earlymaturing stem cell progeny with or without maintenance of thisexpression level change in late maturing cells. Interestingly,up-regulation of p53 expression has been detected predominantly duringearly differentiation events of mouse embryogenesis and decreased duringterminal differentiation (Schmid et al, “Expression of p53 during mouseembryogenesis”, Development, 113:857-865 (1991)). In addition, WT-1 isexpressed in mesenchymal stem cells and immature epithelial cells duringvertebrate development and largely down-regulated in maturing progeny(Kent et al, “The evolution of WT1 sequence and expression pattern inthe vertebrates”, Oncogene, 11: 1781-1792 (1995)). Similarly, RB-1expression is also confined to distinguishable-compartments indeveloping adult tissues (Cordon-Cardo et al, “Expression of theretinoblastoma protein is regulated in normal human tissues”, Am. J.Path., 114:500-510 (1994)). Namely, cells of stratified epitheliadisplay pRB expression in maturing suprabasal layers whereas the basallayer shows lower expression levels (Cordon-Cardo, C., and Richon, V. M.Expression of the retinoblastoma protein is regulated in normal humantissues, Am. J. Path., 114:500-510 (1994)). Further elucidation of thedownstream molecular elements leading to TS-induced changes in cellgrowth kinetics should confirm whether the action of certain TS'sinvolves a one step switch in mitotic mode.

2. Are TS's Initially Epigenetically Down-Regulated in Cancer Cells?

If TS's do normally play a role in maintaining the division kinetics ofrenewing stem cells in maturing mammalian tissues, then it would followthat TS's and/or their pathways should be largely down-regulated in stemcells during the majority of their cell cycle. Since by the OSES modelcancer cells inherit the expression profile of stem cells en bloc, geneswhich are natively down-regulated in stem cells (e.g., TS's) should becorrespondingly unexpressed in cancer cells. While direct evidence forsuch a proposal remains to be demonstrated, there are a variety of datasuggesting that expression of certain TS's and/or their pathways may bealtered and in some cases down-regulated in certain tumors without anobvious mutational cause for this. For example, there is a subset oftumors in which epigenetic down-regulation of a wild type TS has beenoffered as a mechanism to explain the enigmatic presence of loss ofheterozygosity (LOH) of a TS accompanied by a seemingly wild typeremaining allele (e.g., in the case of TP53, WT-1, and RB-1) (Feinberg,“Genomic imprinting and gene activation in human cancer”, Nat. Genet.,2:110-113 (1993)). While definitive evidence for altered expression ofwild type WT-1 or RB-1 remains to be demonstrated in these cases,altered p53 protein expression without detectable concomitant somaticTP53 mutation has indeed been described in a variety of human tumorspecimens of hematologic, colonic, and pituitary origin. While theseresults had originally been attributed simply to insufficient TP53sequence analyses, more rigorous genomic sequencing coupled with thereproducibility of these findings by several independent laboratoriesindicates that altered regulation of wild type TS expression, at leastin the case of TP53, is an actual cancer-related phenomenon for whichalternative mechanisms might be required to adequately explain (Ohue etal, “A Frequent Alteration of p53 Gene in Carcinoma in Adenoma ofColon”, Cancer Res., 54:4798-4804 (1994); Kaklamanis et al, “p53Expression in Colorectal Adenomas”, Am. J. Path., 142:87-93 (1993));Levy et al, “p53 gene mutations in pituitary adenomas: rare events”,Clin. Endocrinology, 41:809-814 (1994); Greenblatt et al, “Mutations inthe p53 Tumor Suppressor Gene; Clues to Cancer Etiology and MolecularPathogenesis”, Cancer Res., 54:4855-4878 (1994); Ueda et al, “Functionalinactivation but not structural mutation of p53 causes liver cancer”,Nat. Genet., 9:4147 (1995)). Some other possible explanations offeredfor such findings within the confines of a conventionalmutation-selection model include altered splicing or promoter-regionmutations causing changes in wild type TP53 expression. While thesehypothetical mechanisms certainly remain viable until adequately tested,they do not appear capable of accounting for all cases of cancer-relatedexpression changes in wild type TS's. Namely, while down-regulation ofwild type BRCA-1 mRNA has been reported in a number of sporadic humanbreast cancers, close inspection reveals that both differential splicingand promoter region mutations are unlikely explanations for suchfindings. Accordingly, it has been proposed that mutational inactivationof a BRCA-1 regulatory gene might be another conceivable explanation,within the boundaries of conventional mutation-selection, for theseseemingly enigmatic data (Thompson et al, “Decreased expression ofBRCA-1 accelerates growth and is often present during sporadic breastcancer progression”, Nat. Genet., 9:444-450 (1995)). However, thesefindings are precisely those to be expected by an OSES model whereincertain TS's are initially epigenetically down-regulated in cancercells.

One potential problem with the conventional model which is forced toinvoke mutational inactivation of unexamined loci (in this caseregulatory genes) prior to actually demonstrating its presence, as withthe case of implicating a MP process, is that such notions are virtuallyimpossible to wholly disprove. Accordingly, other models should at leastbe considered. Namely, epigenetic down-regulation might be anotherexplanation for the finding of an unexpressed wild type TS in certaintumors. This is not the first time such a proposal has been offered(Feinberg, “Genomic imprinting and gene activation in human cancer”,Nat. Genet., 2:110-13 (1993)). Namely, reports that alterations in p53protein accumulation present at the onset of certain early sporadiccolonic neoplasms prior to the occurrence of any major mutational damagehas led some to invoke a non-mutational mechanism for these p53expression level alterations (Greenblatt et al, “Mutations in the p53Tumor Suppressor Gene: Clues to Cancer Etiology and MolecularPathogenesis”, Cancer Res., 54:4855-4878 (1994)). While an epigeneticetiology for a change in TS expression could in theory reflect either ageneralized neo-Darwinian or an OSES process, an OSES model would be thefavored explanation if epigenetic down-regulation of a given TS wasdemonstrated in both tumor cell as well as stem cell of the same tissuetype, i.e., circumstantial evidence for direct inheritance. Even moreconvincing for an OSES model would be demonstration that certain TSmutation's had occurred subsequent to an initial epigeneticallydown-regulated state.

3. Does Mutational Inactivation of TS's in Cancer Cells Occur Subsequentto an Initial Epigenetically Down-Regulated State?

By the OSES model, cancer cells are susceptible to reversion via theinducing effects of local “differentiation signals”. Therefore, thosecancer cells situated at the periphery of a growing neoplastic massshould be most subject to differentiation/reversion and thus more proneto selection for mutant differentiation-defective outgrowths than theirmore centrally-located counterparts. In this manner, as mentioned, thosemutants situated at the periphery of a tumor might effectively “shield”a smaller subpopulation of centrally-located cancer cells fromdifferentiation induction, (i.e., selection) thereby allowing such cellsto preserve their genetic integrity and thus harbor the hard evidencefor an OSES origin for cancer. Accordingly, there may exist only a smallwindow of opportunity during the initial phases of spontaneous tumordevelopment and only a small centrally-located stem line of cells forwhich to demonstrate experimentally the prediction by the OSES modelthat epigenesis precedes mutagenesis.

There is at least one particular experiment which may demonstrate such arelated phenomenon. Namely, in liver cancers derived from transgenicmice expressing a single hepatitis B virus (HBV) transgene, the encodedviral protein, HBx, binds p53 protein and prevents its entrance into thenucleus thereby effectively epigenetically down-regulating the p53pathway. Interestingly, however, is that while analysis of tumor cellDNA derived from early lesions reveals no evidence of p53 mutation (afinding consistent with an epigenetic mechanism for itsdown-regulation), more advanced lesions do display a small number ofcells with acquired TP53 base substitutions, a process which apparentlyoccurred subsequent to an initially epigenetically down-regulated TP53state (Ueda et al, “Functional inactivation but not structural mutationof p53 causes liver cancer”, Nat. Genet., 9:4147 (1995)). Byconventional models, it is not abundantly clear why epigeneticinactivation of a cancer-related gene would be followed by itsmutational inactivation during tumor “progression”, i.e., why wouldthere be selection for mutational inactivation of a gene which wasalready (epigenetically) inactivated? One potential explanationconsistent with conventional models is that epigenetic inactivationprovides an initial growth advantage to cells but is not as absolute asmutational inactivation (Ueda et al, “Functional inactivation but notstructural mutation of p53 causes liver cancer”, Nat. Genet., 9:41-47(1995)). However, this idea like that of a MP is difficult to disprove.Moreover, that transdifferentiation of one mature cell type to anotheris not a common phenomenon in mammals is evidence that epigeneticalterations leading to cell fate decisions is a relatively permanentprocess in no need of accompanying genomic is alterations (Rubin, H.,“Cancer as a Dynamic Developmental Disorder”, Cancer Res., 45:2935-2942(1985)). Additional transgenic experiments of the type mentioned aboveexamining other TS's should corroborate preliminary findings thatmutational inactivation of a TS can follow epigenetic inactivation. Inaddition, more intensive molecular analyses of tumor specimens at theRNA level (e.g., by in situ reverse transcription polymerase chainreaction) should better elucidate the temporal relationships betweencertain mutational and epigenetic events during the early phases ofcarcinogenesis.

As presented above, there exists a patchwork of preliminary evidencethat the expression of certain TS's and/or their pathways may follow thepredictions of the OSES model. For example, there is evidence that T53functions in adult tissue development as a provoker of differentiationpossibly via a cell cycle-coordinated induction of an asymmetric mitoticprogram in stem cells (Sherley et al, “Expression of the wild type p53antioncogene induces guanine nucleotide-dependent stem cell divisionkinetics”, Proc. Natl. Acad. Sci. USA, 92:136-140 (1995)). It followsfrom these data that TP53 should be largely down-regulated in the nativenon-dividing state of stem cells. Moreover, there are also findings thatalterations in TP53 expression may be present in tumor cells withoutevidence of a somatic mutation (Ohue et al, “A Frequent Alteration ofp53 Gene in Carcinoma in Adenoma of Colon”, Cancer Res., 54:4798-4804(1994); Kaklamanis et al, “p53 Expression in Colorectal Adenomas”, Am.J. Path., 142:87-93 (1993); Levy et al, “p53 gene mutations in pituitaryadenomas: rare events”, Clin. Endocrinology, 41:809-814 (1994)). Whetherthese data reflect an initial inherited down-regulated state for TP53with subsequent up-regulation and deductibility during cancer celldifferentiation rather than a neo-Darwinian “progression” of mutantforms may warrant more intensive molecular dissection to determinewhether wild-type TP53 RNA levels positively correlate with decreasinghistopathologic grade of a given tumor portion, as would be predicted byan OSES model. In addition, the preliminary evidence that TP53 mutationcould potentially follow an initial epigenetic down-regulated TP53 statein some tumors, as illustrated by the HBV transgenic mouse model (Uedaet al, “Functional inactivation but not structural mutation of p53causes liver cancer”, Nat. Genet, 9:41-47 (1995)), indicates that thisparticular prediction of the OSES model is possible. Are other TS's forwhich evidence suggests a down-regulated state in stein cells (e.g.,WT-1, RB-1) and an epigenetically down-regulated state in cancer cells(e.g., BRCA-1) also mutationally inactivated only after an initialepigenetically inactivated state? (Cordon-Cardo et al, “Expression ofthe retinoblastoma protein is regulated in normal human tissues”, Am. J.Path., 114:500-510 (1994); Marquis et al, “The developmental pattern ofBrca1 expression implies a role in differentiation of the breast andother tissues”, Nat. Genet., 11:17-26 (1995); Thompson et al, “Decreasedexpression of BRCA-1 accelerates growth and is often present duringsporadic breast cancer progression”, Nat. Genet., 9:444-450 (1995)).Corroboration of this latter point with future experiments would bestrongly supportive of an OSES model for cancer development anddifficult for conventional models to reconcile.

Recent neo-Darwinian models have invoked several mechanisms including aMP, mutational inactivation of as yet unidentified/undefined geneticloci (e.g., regulatory genes or promoter regions), and most recently theconcept of epigenetic down-regulation not being as absolute asmutational inactivation in order to account for a variety of enigmaticcancer-related findings. However, based on the foregoing, the OSEScancer model provides a more reasonable and logical explanation ofcarcinogenesis which obviates the inaccuracies and ambiguitiesassociated with prior neo-Darwinian cancer models. Moreover, asmentioned, the OSES model can better account than can conventionalmodels for instances of 1) elevated transformation rates not explainedby somatic mutation alone, 2) tumor differentiation and reversibility,and 3) the presence of mutations in both early and late human neoplasticlesions surprisingly without evidence of selection.

OSES-Based Cancer Diagnostic Methods and Therapies

Thus, the present invention provides novel methods for the detection andfor the treatment of cancer which are based on the OSES model. The OSESconcludes that a clandestine slow-growing relatively mutationally-sparedfounder line, termed the cancer stem line, (created via a one-stepepigenetic switch from asymmetric to symmetric stem cell mitosis andthen persistent throughout the life of the tumor) exists within tumorsand is responsible for fueling tumor immortality. Like its normal stemcell counterpart (from which it is derived and to which it shares manyfeatures), the cancer stem line is slow-growing and rears a transitpopulation of highly proliferative (often mutant) progeny which makes upthe bulk of the resulting mass. This transit population in tumors, likein normal tissues, likely has limited proliferative potential (versusits progenitor cancer stem line population which is immortal).

Previous conventional cancer models have focused on the fast-growinghighly mutant cell population in tumors. This population is widelybelieved to have arisen by an evolutionary process involving stepwisemutation-selection at the cellular level. However, such conventionalmodels have not considered the presence of a cancer stem line (of thetype proposed by the OSES model)—a significant difference which rendersthe OSES model and its novel detection methods and therapies for cancerclearly, distinguishable from those of conventional cancer models.

Thus, based on the OSES model, the present invention provides 1) novelmethods of cancer detection which may detect cancer at an earlier stagethan conventional detection methods, as well as 2) novel cancertherapies which may be more apt to cure cancer (i.e. by eradicating itsimmortal portion) and thereby less prone to relapse than conventionaltherapies. Conventional methods of cancer detection rely on the presenceof relatively large numbers of proliferative cancer cells (e.g., largenumbers of cells are necessary to allow detectability by conventionalmethods such as physical exam, radiologic studies, blood tests, etc.)thereby making cancer detection possible only at a relatively laterstage than would be possible by detection methods derived from the OSESmodel which seeks to identify a small cancer stem line sub-populationcreated early in tumorigenesis. Similarly, conventional cancer therapies(e.g., chemical chemotherapies, irradiation, immunotherapies,experimental gene therapies) are designed to target highly proliferativecells, thereby sparing a cancer stem line (of the type described by theOSES model) and thus making these types of therapy prone to cancerrelapse. OSES-based therapeutic regimens will target the cancer stemline and thus may be less susceptible to relapse and more prone to truecancer cure. Such OSES-based therapies could be used in combination withclassical chemotherapies to accelerate diminution of tumor burden whilealso targeting the clandestine cancer stem line.

Novel Therapies Provided by the Invention

As discussed, it is widely accepted that a cancer cell arises via amulti-step progression from a normal cell to an increasingly“cancer-like” (i.e. pre-cancerous) cell leading ultimately to afull-fledged cancer cell with malignant capability. This process, as iscurrently described, is driven by neo-Darwinian evolution whereby thoseincreasingly “cancer-like” cells with the most advantageous mutations(e.g., growth-promoting ones) undergo natural selection and expansionover their less “fit” competitors. In this manner, multiple rounds ofmutation followed by selection lead ultimately to a malignant cancercell which is both highly proliferative and highly mutated. It is thisgenerally accepted paradigm for cancer development by which conventionalcancer chemotherapies are based. Namely, classic cancer chemotherapies(e.g., alkylating agents such as cyclophosphamide, anti-metabolites suchas 5-Fluorouracil, plant alkaloids such as vincristine) as well asirradiation-based therapies have been employed in cancer treatmentbecause of their adverse effects on cellular growth and DNA replicationand thus toxicity to highly proliferative (cancer) cells. In a similarmanner, certain immunotherapies as well as newer experimental genetherapies have also focused on highly proliferative (and often mutant)cells as the prime targets for cancer treatment. However, while some ofthese mentioned therapies (i.e. namely the more conventional chemicalchemotherapies and irradiation regimens) can induce remission (andoccasionally cure) in a subset of cancer types, such therapies do notprolong survival for the majority of patients with cancer. Thisunfortunate fact is often attributed to technical reasons such asinability to give large enough doses (e.g., due to toxicities), or tothe development by cancer cells of resistance to therapy. However, itshould be noted that such therapies could also be of limited clinicalbenefit for other reasons—namely, if the neo-Darwinian paradigm forcancer development (i.e. the basis of conventional as well as newertherapies) were flawed then so too would therapies wholly based on thismodel.

As an alternative to conventional models, the OSES model, as mentioned,argues that a tumor does indeed largely consist of highly proliferativeand mutated cells (in agreement with conventional models) but makes theadditional novel proposal that tumors are also (in contradistinction toconventional models) fueled by a clandestine founder sub-population ofslow-growing relatively mutationally-spared cells (termed a “cancer stemline”) which is no formed by neo-Darwinian cellular evolution but ratherby a “quantum leap” of sorts (i.e. a one-step switch in mitotic mode).In this manner, the cancer stem line fuels tumor growth by acting as itsimmortal founder line which rears a much larger (and thus more readilyidentifiable) population of highly proliferative mutated progenycells—i.e., that proliferative mutant population of cells which asmentioned has been the focus of classic chemotherapies, immunotherapies,as well as newer gene therapies. Accordingly, by the OSES model,therapies based on the conventional neo-Darwinian model of cancer (byvirtue of their design to target highly proliferative and/or mutantcells) while destroying proliferative mutant cells (i.e. progeny of thecancer stem line which themselves are largely mortal) fail to eradicatethe slow-growing relatively mutationally-spared cancer stem lineresponsible for fueling tumor immortality. Thus, while suchconventionally-based treatments may induce clinical remission bytransiently reducing overall tumor cell burden, by the OSES model thesetherapies will ultimately fail to cure cancer in most cases because of afailure of eradicate the cancer stem line which possesses the potentialto repopulate the tumor thereby causing relapse (FIGS. 2A and 2B).

In very simple terms, this scenario would be analogous to transientlykilling roaches with Raid® without eradicating their eggs (i.e. stemline) which would need to be targeted by different means (e.g., Combat®)to prevent relapse. That is, conventional therapies use Raid®-likeregimens, while OSES-based therapies would use Combat®-like regimens.This is not to say that the conventional manner (using classicchemotherapies or newer gene therapies) of destroying the highlyproliferative mutated cells (which comprise a large majority of thecells within a given tumor) has no clinical utility—it obviously does bysignificantly lowering the tumor cell burden which at times can produceclinical remission—but by the OSES model a clandestine cancer stem linestill remains untreated (FIGS. 2A and 2B).

Accordingly, it follows from the OSES model that it is thenot-yet-appreciated cancer stem line sub-population which is responsiblefor fueling tumor immortality, a population which escapes classictherapies which themselves are based on a flawed but widely acceptedneo-Darwinian paradigm wherein the key cancer cells which should betargeted by therapy are those which are highly proliferative and highlymutated. This flaw extends not only to classic chemical therapies andirradiation modalities but also to other newer and more experimentalcancer therapies including tumor-directed monoclonal antibodyimmunotherapies which have targeted novel antigens present on highlyproliferative (often mutant) cancer cells as well as certain newerexperimental gene therapies designed (with the conventional paradigm inmind) to correct or override those mutations accrued by cancer cellsduring their evolution, e.g., by but not exclusive to either 1)destroying mutant cells, as attempted by certain preliminary DNA or RNA(e.g., antisense or ribozyme) therapies “custom-designed” to lethallytarget certain specific cancer-related mutant genes or their geneproducts, or 2) “correcting” mutant cells, as demonstrated by recentpreliminary gene-directed therapies designed to “override” certaincancer-related mutations via insertion of wild-type versions of thesegenes into cancer cells (Karp, et al, “New Directions in MolecularMedicine”, Cancer Res., 54:653-665 (1994), Kashani-Sabet et al,“Suppression of the Neoplastic Phenotype in Vivo by an Anti-rasRibozyme”, Cancer Res., 54:900-902 (1994)). However, while theseconventionally-based methods of cancer treatment may eradicate asignificant proportion of the tumor mass by destroying the highlyproliferative and mutant neoplastic population thus potentiallyresulting in clinical remission, in time the tumor may recur at the sameor different site(s) for the same reasons that classic chemicalchemotherapies often fail. A number of reasons for tumor relapse andchemotherapeutic failure have been offered within the conventionalparadigm. These include insufficient chemotherapeutic dosage (limited byonset of significant side effects), and/or emergence of cancer clones(e.g., to mutants) which are resistant to therapy. By the OSES model,the emergence of mutant cancer clones (which may or may not be immortal)may occur in concert with the presence of an underlying cancer stemline, the latter of which has yet to be properly targeted by therapy(FIG. 2A).

The OSES model, as mentioned, offers an alternative explanation forrelapse wherein a clandestine slow-growing mutationally-spared cancerstem line, not targeted by conventional therapies, is the immortalfounder line of the tumor which may in time gradually regrow the tumormass. As mentioned, since such a cancer stem line shares growth kineticswith normal stem cells (from which it is directly derived via a one-stepepigenetic switch in mitotic mode), i.e. that being a slow-growingimmortal cell population which rears progeny cells which themselves arefast-growing and mutant, therapies designed to target fast-growingmutant cells will spare the cancer stem line. Accordingly, noveltherapies are needed which specifically target this cancer stem line.This can be accomplished by, but is not exclusive to, newly designedtherapies which 1) are specifically cytotoxic to the cancer stem line,e.g., via targeting specific surface antigens, cellular contents, othergene products present in the cancer stem line by immune or otherdirected cytotoxic therapies “tailor-made” to target the cancer stemline, or 2) force the cancer stem line to switch, permanently orotherwise, from exponential (symmetric) malignant growth to a less“dangerous” arithmetic (asymmetric) growth pattern, or 3) extinguish thecancer stem line by forcing it to permanently adopt a terminaldifferentiation or apoptotic (i.e. programmed cell death) program.Moreover, it is a goal of such OSES-based cancer therapy to spare normalstem cells of significant therapy-associated toxicity.

Since the malignant stem line is equivalent to symmetrically-dividingstem cells, therapies for eradication of such a cell population willinclude, but not be exclusive to:

I. immunotherapy directed at normal adult stem cells antigens since suchantigens will largely be present on a cancer stem line derived from stemcells of a given tissue type having been inherited en bloc via aone-step epigenetic switch) (FIG. 3A); and/or

II. induction of a switch from symmetric to asymmetric mitosis in thecancer stem line by activating or suppressing positively ornegatively-acting factors, respectively, which are normally involved incontrolling this switch in normal stem cells thereby effecting a changefrom exponential to arithmetic tumor growth (FIG. 3B); and/or

III. induction in the stem line of terminal differentiation or apoptosis(i.e. programmed cell death) by causing a switch from a symmetricproliferative mitotic program which normally responds todifferentiation-inducing signals or starvation by assuming an asymmetricquiescent growth phase (thereby retaining a proliferative capability forlater use) to one that switches to a symmetric but terminaldifferentiation or apoptotic program whereby a proliferative capabilityis irreversibly lost (FIG. 3C). By this method, mitotic mode isuncoupled from differentiation—a process which occurs to various degreesin lower eukaryotes.

These are three examples of how the cancer stem line could be targetedand contained for the treatment of cancer. Therapies to treat cancerbased on the OSES model include such methods but are not exclusive tothem and may involve additional modalities as newer technologies aredeveloped and additional information on the molecular mechanisms of howa stem line (normal or neoplastic) behaves becomes available which willprovide new heretofore unknown cancer stem line targets. The mentionedtherapies are described in greater detail below, but are not intended tobe exhaustive.

I. Targeting A Cancer Stem Line Via Novel Immunotherapy

Cancer immunotherapy in the past has been designed to target antigenswhich are mutated and/or present on highly proliferative cancer cells,which by the OSES model would constitute antigens present on cancer stemline progeny rather than the cancer stem line itself. Accordingly, bythe OSES model, classic immunotherapy (like classic chemotherapies andirradiation treatments) is similarly flawed because while it may destroythe bulk of cancer stem line progeny, it will not target the underlyingcancer stem line which fuels tumor immortality.

As mentioned, according to the OSES model, cancer cells aresymmetrically-dividing stem cells. Therefore, certain antigens presenton normal stem cells will also be present on cancer cells for “neutral”reasons (i.e. because of en bloc inheritance) rather than for reasons of“selection”. In addition, antigens shared by both cancer cells and stemcells should also to some extent be present on embryonic progenitorcells from which adult stem cells are derived (due also to inheritance).Accordingly, as embryonic progenitor cells, stem cells, and cancer cellsfrom the same tissue of origin will share certain surface antigens(which may subsequently be lost during cell differentiation and thus notreadily detectable in adult tissues), identification of such sharedantigens for use as potential targets for immunotherapy for cancer(i.e., to target the cancer stem line) should be sought. This can beassisted via study of embryonic or adult stem cells, for the purpose ofidentifying cell surface antigens present on these non-cancerous celltypes, which may be technically easier to isolate and characterize thanthose on cancer stem line cells themselves. A proportion of suchantigens would then, according to the OSES model, be presumed to bepresent on a cancer stem line derived from that particular tissue andthus worthy of therapeutic targeting. This method contrasts with classicimmunotherapies which have not targeted native stem cell antigens butrather antigens present on highly proliferative (often mutant) cancercells, which by the OSES model is a cell population that does notrepresent the immortal population which needs to be most aggressivelytargeted. In support of the above OSES-derived proposal that certainwildtype cell products (e.g., cell surface antigens) may be shared byembryonic cells and adult stem cells along with cancer cells from thatparticular tissue type, there is evidence that some tumor cell types(both of hematopoietic as well as solid origin) share expression ofisoforms of certain fetal stage-specific genes including in some casesembryonic cell-surface antigens with their normal stem cell counterparts(Sachs, “Cell Differentiation and Bypassing of Genetic Defects in theSuppression of Malignancy”, Cancer Res., 47:1981-1986 (1987); Hall,“Stem Cell Is a Stem Cell Is a Stem Cell”, Cell, 33:11-12, (1983); Sigalet al, “The liver as a stem cell and lineage system”, Am. J. Physiol.,263:G139-G148 (1992)). For example, certain cell surface antigens (e.g.,SSEA antigen family) detected on murine germ cell-derived tumors havealso been detected on adult oocytes (i.e. germline stem cells) as wellas on 4-8 cell mouse embryos (Hall, “Stem Cell Is a Stem Cell Is a StemCell”, Cell, 33:11-12, (1983)). Moreover, expression of several cellsurface antigens as well as other primitive gene products includingalpha-fetoprotein (AFP) and IGF-2 have been detected in hepatic tumorcells, adult stem cells of the liver, as well as in fetal liverindicating shared gene expression by these temporally distinct butrelated cancerous and non-cancerous cell types (Sigal et al, “The liveras a stem cell and lineage system”, Am. J. Physiol., 263:G139-G148(1992)). While conventional models might attribute such findings todedifferentiation of an adult cell to a more primitive form, the OSESmodel as mentioned argues that such similarities in gene expressionbetween cancer cells, adult stem cells, and embryonic cells is due toinheritance and not to dedifferentiation or selection. In this manner,the OSES model seems more parsimonious than conventional ones in that itneed not be forced to invoke selection (as conventional models are) toexplain the presence of surface antigens which appear for all intentsand purposes to be “neutral” (i.e. without any selective benefit) aconcept which has posed somewhat of a problem for conventional models.

It follows from this idea that if a cancer stem line shares expressionof certain gene products (e.g., certain cell-surface antigens) withadult stem cells and embryonic cells of its tissue type of origin thenimmune-directed targeting of certain native antigens present onembryonic progenitors and stem cells from the same tissue lineageshould, by the OSES model, target the cancer stem line. By contrast,cancer immunotherapies in the past have been directed toward antigenspresent on the highly proliferative (and often mutant) population ofcancer cells and thus, in a similar manner to conventional chemicalchemotherapies, will not eradicate the immortal cancer stem line whosesurface antigens are likely non-mutant and distinct from those expressedby their proliferating and/or differentiating progeny.

By the OSES model, suitable cancer stem line antigens to be targeted bymonoclonal antibodies may be identified by, e.g., cloning adult stemcells of various tissue types in order to determine their expressionprofiles and complement of cell surface antigens for a particular tissueand assuming that a proportion of these cell products will also bepresent in a cancer stem line derived from these stem cells (because ofen bloc inheritance). Cloning of adult stem cells of hematopoieticorigin is a technique previously described and now done routinely bythose skilled in the art (Uchida et al, “Heterogeneity of hematopoieticstem cells”, Curr. Opin. Immunol., 5:177-184 (1993)). There has now alsobeen some preliminary success in isolating stem cells ofnon-hematopoietic tissues, namely gastrointestinal epithelia (Gordon etal, “Differentiation and self-renewal in the mouse gastrointestinalepithelium”, Curr. Opin. Cell Bio., 6:795-803 (1994)). Improvedpurification methods to isolate (non-hematopoietic) stem cells fromtheir non-stem cell neighbors will, possibly aided by separationtechniques that focus on gene products differentially expressed in stemand non-stem cells as targets for separation (e.g., data gleaned fromboth normal and virally-infected lineage studies), will undoubtedly helpin the identification of stem cell-specific gene products (e.g.,integrins as seen in epidermal stem cell systems) (Jones et al, “Stemcell patterning and fate in human epidermis”, Cell, 80:83-93 (1995)),which will also include products which are cell surface antigens (Sigalet al, “The liver as a stem cell and lineage system”, Am. J. Physiol.,263:G139-G148 (1392); Villarreal, Relationship of Eukaryotic DNAReplication to Committed Gene Expression: General Theory for GeneControl, Microbiol. Rev., 512-542 (1991)). In other words, as more stemcell-related data become reported so too will the number of possibleantigens for which to target a cancer stem line derived from a giventissue type by these novel methods.

Monoclonal antibody construction specific to hematopoietic stem cells isa technique which has been previously well-described (Uchida et al,“Heterogeneity of hematopoietic stem cells”, Curr. Opin. Immunol.,5:177-184 (1993)). Although a technique not yet as perfected, cloning ofnon-hematopoietic stem cells and construction of monoclonal antibodiesspecific to them has also had some preliminary success (Hall, “Stem CellIs a Stem Cell Is a Stem Cell”, Cell, 33:11-12, (1983)). Accordingly, bythe OSES model, perfection of this technique can then be used to target(via immunotherapy) a cancer stem line arising from that particular stemcell type. Construction of cell type-specific monoclonal antibodiesinvolve methods well-known to those skilled in the art. In this manner,antibodies designed to be non-tolerant of antigens native to adult stemcells (and/or their precursor embryonic cells) will selectively home inon stem cells which are no longer environmentally sequestered (i.e., acancer stem line). It should be noted that several monoclonal antibodieshave already been constructed against non-hematologic tissues includingmurine embryonic cells (Hall, “Stem Cell Is a Stem Cell Is a Stem Cell”,Cell, 33:11-12, (1983)). Their efficacy as potential therapeutic agentsfor cancer (a novel idea which follows from the OSES model) awaitsdemonstration. Evidence that certain antigens may be shared by relatedbut independent developing tissue types may also enable certainmonoclonal antibodies made to one embryonic/stem cell type to be used totreat more than one cancer cell type thereby expediting assessment ofthe efficacy of this novel OSES-derived method for treating cancer(Hall, “Stem Cell Is a Stem Cell Is a Stem Cell”, Cell, 33:11-12,(1983)). Thus, the OSES method of immune-directed targeting ofneoplastic cells differs from previous cancer immunotherapies the latterof which have targeted mutant tumor antigens or antigens present onproliferating or differentiating stem line progeny rather than (asrecommended by the OSES model) on wild type antigens normally expressedby embryonic progenitor and adult stem cells, i.e. antigens presumed toalso be present on a cancer stem line.

It should also be noted that some previously described cancerimmunotherapeutic regimens have attempted to augment an endogenousimmune response to cancer. However, by the OSES model, this method issimilarly flawed because antigens native to embryonic and adult stemcells may not normally be viewed as foreign by the immune system (sinceimmune progenitor cells are likely exposed to such native embryonicantigens prior to adoption of immunologic tolerance) and thus the immunesystem might be tolerant to antigens expressed on a cancer stem line andtherefore not attack it. This idea is unique to the OSES model. In thismanner, simply augmenting the native immune response to a tumor by anyof a variety of previously described means would not be expected toresult in eradication of a non-foreign-appearing cancer stem line. Onthe other hand, promotion of cytotoxicity to this otherwise“normal-appearing” but pathogenic cancer stem line (such as byengineering monoclonal antibodies-targeting it, as described above)would, by the OSES model, result in its demise. In addition, ex vivoinduction of embryonic/stem cell antigen non-tolerance in progenitor(i.e., “impressionable”) immune cells followed by their reintroductioninto a patient with cancer of that particular cell type would be anotherway by the OSES model to effect cancer stem line immune-directedcytotoxicity. Ex vivo therapies used for other reasons have beendescribed and are well-known by those skilled in the art.

Once monoclonal antibodies to stem cells are obtained, they may be usedto produce immunotherapies by conventional methods. For example, themonoclonal antibody may be attached directly or indirectly to atherapeutic moiety, e.g., a therapeutic enzyme, chemotherapeutic agent,cytotoxin, lymphokine, cytokine, radionuclide, anti-metabolite, orderivatives thereof, or may be used alone to stimulate native immunesystem attack. Alternatively, the antibody may be used in pre-targetingtherapies which in turn administer an antibody—(ligand or anti-ligand)conjugate which binds to targeted cells, followed by administration of a(ligand or anti-ligand) therapeutic moiety conjugate. Such methods areoften favored over administration of antibody-therapeutic agentconjugates as they may reduce non-specific cytotoxicity. Also, it isdesirable that the antibody be substantially non-immunogenic in thetreated subject. This may be accomplished by chimerizing or humanizingthe monoclonal antibody, or producing a single chain version thereof. Inaddition, antibody fragments, such as Fab fragments are less immunogenicbecause of their smaller size.

Methods for administering antibodies are well known in the art andinclude parenteral modes of administration such as intramuscular andintravenous injection, as well as systemic routes of administration,e.g., oral, intranasal etc. Generally, an antibody or antibody conjugatewill be administered in combination with a pharmaceutical carrier orexcipient, and in conjunction with moieties that preserve the stabilitythereof, e.g., buffers, and compounds which maintain protein stability.The dosage amount will vary within wide limits. Generally, it will varyfrom about 0.001 mg to 10 mg/Kg body weight.

However, a possible side effect of OSES-based treatment of a cancer stemline is potential toxicity to normal stem cells. This will dependlargely on whether normal stem cells, by virtue of their environmentalsequestration, are spared of stem/cancer cell immune-directed attack. Inother words, it is possible that stem cells within themicro-environments of solid tissues are not accessible toimmune-directed attack thereby reducing the side effects of such noveltherapy. If normal stem cells are susceptible to monoclonal antibodytargeting, then at alternative approach whereby such monoclonalantibodies are used solely for detection of a cancer stem line ratherthan eradication thereof (e.g., via tagging to a benign identifiablemarker such as fluorescence, as will be discussed later in the detectionsection) such that detection and localization could lead to early localconventional modes of therapy (e.g., irradiation or surgical removal) ofwhat would have been undetectable cancer cells. This method would bewithout adverse effects to normal stem cells (other than temporarybenign fluorescence). Moreover, successful removal of a cancer stem linemight allow further characterization of its expression profile and cellsurface antigens so as to determine distinguishing features from itsnormal stem cell counterparts to which to design more specificimmunotherapy so as to avoid targeting normal stem cells if morespecific therapeutic targeting is warranted.

EXAMPLE 1

A patient is found to have breast cancer by conventional detectiontechniques (e.g., mammogram and biopsy). Monoclonal antibodies designedto bind specific antigens present on the surfaces of normal mammarygland epithelial stem cells, also possessing an attached therapeuticmoiety (e.g., cytotoxin), are delivered intravenously to the patient.This therapeutic monoclonal antibody will seek out and destroy a cancerstem line fueling the breast tumor while largely sparingenvironmentally-sequestered normal mammary epithelial stem cells (seeFIG. 3A). Efforts td limit this toxicity further are discussed in thefinal section, (III).

II. Forcing a Cancer Stem Line to Undergo Asymmetric Mitosis

An alternative tactic to immunotherapy for containing a cancer stem lineis to design therapies which (rather than killing stem cells) bringabout the conditions of a normally sequestered stem cellmicro-environment to wayward stem cells (i.e. cancer cells) which inturn should force them to resume asymmetric mitosis and arithmeticgrowth kinetics. Since stem cells are not targeted for death by thisform of therapy, there should be minimal toxicity to normal stem cellswhich presumably are already dividing asymmetrically anyway.

As mentioned, by the OSES model cancer cells are symmetrically-dividingstem cells which, while capable of transiently switching to asymmetricmitosis when induced to do so by local differentiation-inducing signals,will assume an exponential (symmetric) growth pattern when extrinsicdifferentiation-inducing stimuli are lacking (e.g., at morecentrally-located rather than peripheral tumor regions where cancercells are “shielded” of such signals) thereby forming acentrally-located symmetrically-dividing cancer stem line. Accordingly,efforts to contain such a “shielded” cancer stem line via targeteddelivery of therapeutic effectors of asymmetric cancer cell mitosis willlimit stem line size and thus potential numbers of reared progeny. Sucha novel treatment modality for cancer may derive from either: 1) use ofknown differentiation—inducing agents (but to be used in a novel dosingmanner), and/or 2) design of new differentiation-inducing agents (basedon knowledge of native asymmetric mitosis/differentiation machinery). Bythis latter OSES-derived mode of therapy, native localdifferentiation-inducing factors (i.e. those normally present in a stemcell milieu) together with their downstream effectors of asymmetric stemcell mitosis will act as effectors and targets, respectively, fortherapeutic interventional manipulation designed to force a cancer cellto switch from symmetric to asymmetric mitosis.

1) Induction of Asymmetric Cancer Stem Line Mitosis Via Delivery ofKnown Differentiation-Inducing Agents

Coercion of a cancer stem line to divide via asymmetric mitosis can beeffected by various means such as, but not exclusive to, e.g., deliveryof a ligand which upon receptor binding leads to induction of a pathwayleading to differentiation of the cancer stem line (i.e. asymmetriccancer stem line mitosis). This type of treatment modality would betransient however, the duration of which would correlate with thehalf-life of the downstream effects of the delivered ligand. It shouldbe noted that certain differentiation-inducing drugs (e.g., all-transretinoic acid, ATRA) have indeed previously been shown to have clinicalbenefit in cancer therapy, and ATRA is now part of the acceptedtreatment regimen for acute promyelocytic leukemia (Degos, et al“All-Trans-Retinoic Acid as a Differentiation Agent in the Treatment ofAcute Promyelocytic Leukemia”, Blood, 85:2643-2653 (1995)). Otherdifferentiation-inducing compounds such as Hexmethylene bisacetamide,5-Azacytidine, and 1-beta-D-Arabinofuranosylcytosine have also beencited as potential cancer therapies, but their clinical utility awaitsfurther demonstration (Pierce et al, “Tumors as Caricatures of theProcess of Tissue Renewal: Prospects for Therapy by DirectingDifferentiation”, Cancer Res., 48:1996-2004 (1988)). It has beensomewhat enigmatic, by conventional thinking, how cancer cells(presumably having been created via a stepwise evolutionary processinvolving mutation-selection) could reverse such a permanent process(i.e. bypass cumulative genetic derangements) and revert to normal viadifferentiation. By the OSES model, however, cancer cell reversion viadifferentiation is much more readily explained. Namely, the mechanism bywhich differentiation-inducing agents act is via induction of a mitoticswitch in the cancer stem line to asymmetric cancer cell mitosis (i.e.which would appear grossly as tumor differentiation). However, this typeof therapeutic mode, according to the OSES model, would be transientsince after removal of a differentiation-inducing drug the stem linewould resume symmetric mitosis thereby making a treated patient prone torelapse (i.e., re-institution of symmetric cancer cell mitosis) upondiscontinuance of the drug. Accordingly, it would follow from the OSESmodel that more prolonged therapy with differentiation-inducing agentssuch as ATRA (but at lower doses to reduce side effects) would be anovel and more efficacious treatment program than is currently usedbecause it would provide a continual stimulus for a cancer stem line todivide asymmetrically rather than symmetrically. Current dosages of ATRArange from 15-45 mg/m2/d for a recommended treatment duration of 30-45days (Degos, et al “All-Trans-Retinoic Acid as a Differentiation Agentin the Treatment of Acute Promyelocytic Leukemia”, Blood, 85:2643-2653(1995)). While continuous therapy has not been shown to be efficaciousat these dosages, it would follow from the OSES model that lower dosages(e.g., 2-10 mg/m2/d) to avoid toxicities for a prolonged period (e.g.; 6months or longer) should be efficacious. In addition, otherdifferentiation agents (e.g., Hexmethylene bisacetamide, 5-Azacytidine,and 1-beta-D-Arabinofuranosylcytosine) may also provide clinical benefitas a cancer treatment at their respective adjusted low dose andprolonged duration of therapy than is currently suggested.

As directed by the OSES model, in addition to utilizing pre-existingdrugs capable of inducing cell differentiation for the treatment ofcancer (some of which are mentioned above), newer more specificdifferentiation-inducing agents may also be employed. Namely,elucidation of the molecular mechanisms which lead to normal asymmetricstem cell mitosis will allow use of such native factors (as well aspermit their use as templates for the design of “tailor-made” factors)to be exploited as therapeutic effectors of asymmetric cancer stem linemitosis for the treatment of cancer.

2) Induction of Asymmetric Cancer Stem Line Mitosis Via Delivery of NewDifferentiation-Inducing Agents (the Design of which is Based onKnowledge of the Native Asymmetric Mitosis Pathway)

In order to design specific differentiation—inducing cancer therapy thatwill force a cancer stem line to undergo asymmetric mitosis, one mustlook more closely at the molecular mechanisms which bring aboutasymmetric mitosis in normal stem cells in order to decide where and howto intervene in order to constitutively activate such a pathway in acancer stem line. For example, by identifying genes and gene productsrequired for normal asymmetric mitosis, therapeutic interventions can bespecifically designed with high precision (e.g., via activating thoseidentified native factors that drive asymmetric mitosis or,alternatively, blocking native factors that inhibit asymmetric mitosis)to switch a cancer stem line from a symmetric to an asymmetric mitoticprogram thereby altering the growth kinetics of the treated tumor.Toxicity to normal stem cells associated with this type of therapy mayor may not be very significant since normal stem cells are presumablydividing asymmetrically anyway. Of course, efforts to better direct thistype of therapy specifically to a cancer stem line while sparing normalstem cells (as will be discussed) would always be preferred.

The molecular machinery of asymmetric mitosis: Asymmetric mitosis is awidespread process in the animal kingdom that is functionallywell-conserved in organisms ranging from yeast to nematode to insect toman. Mechanistically, it is becoming increasingly evident that cellsutilize some aspect of their pre-existing structural asymmetry toinitiate a mitotic cell division which is also asymmetric in nature(i.e. manifested by unequal segregation of certain intracellular factorsto resulting daughter cells such that these progeny cells assumedifferent fates, e.g., as in mammalian stem cells, one daughter cellrenews pluripotency while the other terminally differentiates).Structural asymmetry can be due to a variety of factors such as aninternal marker (e.g., a bud scar in yeast which marks the site of theprevious cell division, or the site of sperm entry in a nematode zygoteboth of which may provide the cell with a nidus of asymmetry), or anexternal marker (e.g., the apical/basal polarity of an epithelial stemcell as determined by its environment, or the particular membranereceptor site from which the highest concentration of extrinsic signalsare transduced) (Way et al, “Cell polarity and the mechanism ofasymmetric cell division”, Bioessays, 16:925-931 (1994); Lin et al,“Neuroblasts: a model for the asymmetric division of stem cells”, TrendsGenet., 13:33-39 (1997)).

By the OSES model, the asymmetric mitotic pathway in stem cells isinitiated by ligand-receptor binding whereby one or more nativelocally-acting differentiation factors within a stem cell milieu (e.g.,but not exclusive to, gene products of Wnt, Hedgehog, Transforminggrowth factor, Epidermal growth factor) bind to their respectivereceptors (present on stem cells) and induce stem cells to enact anasymmetric mitotic program. Preliminary data from a number of differentorganism suggest that, following ligand-receptor binding, asymmetricstem cell mitosis proceeds via the following general pathway: a)(intrinsic or extrinsic) structural asymmetry in a stem cell is“recognized” (e.g., via inhibition of factors that block itsrecognition) thereby leading to asymmetric assembly of factors whichunequally mobilize b) transcription factors to unequally activate c)genes responsible for determining cell fate whose products becomeunequally apportioned thereby leading to two qualitatively distinctdaughter cells. A number of specific gene products involved at variouspoints of this pathway have been isolated from a variety of organismsTable 1 is a preliminary list of such factors, but is not intended to beexhaustive (Way et al, “Cell polarity and the mechanism of asymmetriccell division”, Bioessays, 16:925-931 (1994)).

It should also be noted that there may be other processes and factors(i.e. not mentioned in Table 1) that act at key points within thispathway to bring about an asymmetric mitotic cell division in normalstem cells. For example, within this preliminary general pathway forasymmetric mitosis (Table 1) there are likely to be points ofconvergence of signals (i.e., “bottlenecks) wherein cellular decisionsare made from a consensus of signals (FIG. 4). Interestingly, there aresome preliminary data that such “bottlenecks” in the mitotic pathwaymay, in part, consist of key cellular events that lead to somaticchromosomal pairing and subsequent exchange events which are inherentlyunequal and thus propagate the initial asymmetry of this entire pathwayleading ultimately to an asymmetric mitotic cell division. This isdiscussed further below.

“Bottlenecks” in the Asymmetric Mitotic Pathway: AllelicPairing/Exchange

As shown in (FIG. 4), the asymmetric mitotic pathway in normal stemcells may contain one or more points of convergence of signals (i.e.“bottlenecks”). It is proposed here that one cellular process which mayrepresent such a “bottleneck” in the asymmetric mitotic pathway is thatof homologous chromosomal pairing and exchange. By this model, followingchromosomal replication in a stem cell, pairing/exchange between one setof homologous chromosomes will mark that daughter cell (receiving theexchanged homologs) for subsequent events leading to the unequalsegregation of factors preferentially to the marked (or unmarked)daughter cell thereby resulting in a difference in fate between daughtercells (i.e. asymmetric mitosis). Such a process is hypothesized to be a“bottleneck” in the pathway because it is presumed to rely on aconsensus of multiple competing ligand-receptor signal transductioneffects such that if a threshold of such signals is met thenpairing/exchange and asymmetric mitosis will proceed, if not thenpairing/exchange will not occur and there will be equal segregation offactors to daughter cells (i.e. symmetric mitosis). What evidencesupports these claims?

It is well known in haploid yeast that allelic pairing/exchange leads toexpression of certain cell fate-determining factors that becomeunequally segregated to resulting daughter cells (i.e. asymmetricmitosis) (Horvitz and Herskowitz, “Mechanisms of Asymmetric CellDivision: Two Bs or Not Two Bs, That is the Question”, Cell, 68:237-255(1992)). In this case, allelic exchange consists of a gene conversionevent at the mating type locus (MAT) which in turn directly leads toup-regulation of a (previously silent) exchanged MAT allele whosedownstream products are asymmetrically apportioned to resulting daughtercells. There is also evidence that a related process (termed“trans-sensing”) occurs in higher diploid eukaryotes. Namely, it hasbeen shown in Drosophila that pairing of homologous alleles in somaticcells can control expression of those paired gene complexes, (Tartof andHenikoff; “Trans-Sensing Effects from Drosophila to Humans”, Cell, 65:201-203 (1991)). In an analogous manner to the yeast system whereallelic pairing affects expression (of one of the paired MAT alleles)via an exchange event at the DNA level it appears that in highereukaryotes (e.g., Drosophila) allelic pairing also affect expression (ofone of the paired alleles) but via an exchange event at the epigeneticrather than genetic level (e.g., which may involve nucleosomes,chromatin, and/or transcription factors). This process appears to also,in a similar manner to DNA exchange events in yeast, require intimatechromosome pairing as well as other downstream recombination-likeprocesses (e.g., heteroduplex formation) suggesting that the processesof genetic and epigenetic exchange may be mechanistically related. Anumber of genes have been isolated which are involved in thetrans-sensing process in Drosophila. Some of these genes include zestewhich alters expression of downstream genes such as the polycomb genes.Other genes involved in this process include white, decapentplegic, andnotch (Wu et at, “The Drosophila zeste gene and transvection” TrendsGenet., 5:189-194 (1989)). While this system awaits further description,there is preliminary evidence that this trans-sensing pathway (involvingzeste-polycomb), like gene conversion in yeast, is also involved inexpression of cell fate determinants that are segregated to daughtercells (Pirrotta, “Transfection and Long-Distance Gene Regulation”,Bioessays, 12: 409-414 (1990)). What about human cells?

There is evidence from a number of sources that homologous chromosomalpairing/exchange occurs in mammalian cells and that alteration of thisprocess leads to certain developmental abnormalities (thereby indicatinga role for this process in normal development). It is proposed here thatthe mechanism by which pairing/exchange functions in mammaliandevelopment is similar to that in yeast and possibly Drosophila, i.e.via affecting expression of one of the paired alleles which in turnleads to expression of downstream cell fate-determinants that areunequally apportioned to resulting daughter cells (i.e. asymmetricmitosis).

Evidence for somatic homologous pairing (and possible exchange ofinformation) in mammalian cells is derived from a variety of sources.For example, there is evidence by interphase cytogenetics thatchromosome pairing normally occurs in mammalian neural cells (Wu,“Transvection, nuclear structure; and chromatin proteins”, J. CellBiol., 120:587-590 (1993)). Interestingly, some of these paired regionscorrespond to regions of homologous recombination in cells from patientswith Bloom's Syndrome. These findings support a role for the wildtypehuman Blooms' syndrome helicase-like gene (BS) in inhibiting DNAexchanges during the presumably normal act of allelic pairing (Tartofand Henikoff “Trans-sensing effects from Drosophila to Humans”, Cell,65:201-203 (1991), Ellis et al, “The Bloom's Syndrome Gene Product IsHomologous to RecQ Helicases”, Cell, 83: 655-666 (1995)).

There are also data that homologous allelic pairing/exchange (i.e.trans-sensing) occurs at additional human loci. For example, confocallaser scanning microscopy and 3-dimensional fluorescence in situhybridization (3D FISH) have revealed that certain homologous loci (inthe chromosome 15q11-13 region) clearly associate with one another insomatic cells (LaSalle et al, “Homologous Association of OppositelyImprinted Chromosomal Domains”, Science, 272: 725—728 (1990)). That suchclose associations at 15q11-13 are followed by exchange ofdevelopmentally-significant information is suggested by several lines ofevidence:

1) This association occurs in well-defined spatial and temporalpatterns.

2) This region is imprinted (i.e. homologous alleles harbor differentepigenetic structure) thereby making an exchange of epigeneticinformation potentially “meaningful” (i.e. in contrast to an exchange ofidentical epigenetic information which would be “meaningless”).

3) Developmental abnormalities arise when pairing in this region isdefective, as seen in Angelman and Prader-Willi syndromes (LaSalle etal, “Homologous Association of Oppositely Imprinted ChromosomalDomains”, Science, 272: 725-728 (1996)). Namely, in both of theseconditions, imprints between homologous alleles at 15q11-13 areidentical which presumably disallows pairing/exchange which in turnleads to aberrant human development.

4) Exchange of epigenetic structure has indeed been shown to followpairing of alleles at 15q11-13, but during meiosis, which suggests thatit might also occur following pairing in other instances, e.g., mitosis(Kelsey and Reik, “Imprint switch mechanism indicated by mutations inPrader Willi and Angelman syndromes”, Bioessays, 19:361-365 (1997)).

There is also evidence for allelic pairing/exchange (i.e. trans-sensing)in the region of human chromosome 11p15, and that such a process directsproper development. Namely, this region like 15q11-13 is also imprinted(thereby making-epigenetic exchange potentially “meaningful”). Inaddition, developmental abnormalities also arise when imprinting (andpresumably paring) is defective in the 11p15 region (i.e. homologousalleles have identical epigenetic structure), as seen in cases ofBeckwith-Wiedemann syndrome (BWS) (Fidler et al, “Trans-sensinghypothesis for the origin of Beckwith-Wiedemann syndrome”, Lancet,339:243 (1992)). Moreover, that exchange of epigenetic structure canindeed occur between homologous alleles in this region is evidenced bydocumentation that a (paternally-derived) imprint was transferred fromone allele of H19 (a gene located in 11p15) to the other (presumablypreceded by allelic pairing) in certain Wilms' tumors (Colot et al,“Interchromosomal transfer of epigenetic states in Ascobolus: Transferof DNA methylation is mechanistically related to homologousrecombination”, Cell, 86:855-864 (1996)). These mentioned findingsindicate that trans—sensing of certain alleles in 11p15 is a necessarycondition for normal development, while inhibition of trans—sensing ofother alleles in this region may be needed to prevent abnormal cellulargrowth.

In a related manner, it has previously been proposed that inhibition ofnative trans-sensing in the regions of human chromosome 3q21, 3q26,16p13, and 16q22 may similarly predispose to abnormal cellular growth(i.e. certain hematologic malignancies, in these cases) (Tartof andHenikoff, “Trans-Sensing Effects from Drosophila to Humans”, Cell, 65:201-203 (1991)).

Trans-sensing has also been implicated in the pathogenesis ofHuntington's disease (HD). Namely, in order to account for thevariability of expression of this dominant phenotype, it has beensuggested that trans-sensing may act between homologous HD alleles (onthe short arm of human chromosome 4) in somatic cells (Laird, “Proposedgenetic basis of Huntington's disease”, Trends Genet., 6:242-247(1990)). The mechanism by which trans-sensing acts in this region islikely similar to that documented for the 11p15 region in somatic cellsand the 15q11-13 in germ cells (and hypothesized for the HD region ingerm cells) where epigenetic structure is exchanged via transfer fromone homologous allele to the other (Colot et al, “Interchromosomaltransfer of epigenetic states in Ascobolus: Transfer of DNA methylationis mechanistically related to homologous recombination”, Cell,86:855-864 (1996); Kelsey and Reik, “Imprint switch mechanism indicatedby mutations in Prader-Willi and Angelman syndromes”, Bioessays,19:361-365 (1997); Sabl and Laird, “Epigene conversion: A proposal withimplications for gene mapping in humans”, Am. J. Hum. Genet.,50:1171-1177 (1992)). Accordingly, it is proposed here that exchange ofepigenetic structure from one paired homologous allele to other resultsin a change in expression of the epigenetically altered allele due to aswitch from either a euchromatin-like to a heterochromatin-likestructure, or vice versa, thereby leading to down or up-regulation,respectively of the affected allele. In this manner, such an expressionchange would lead to induction of downstream cell fate determinantswhich would be unequally segregated to daughter cells. Such a proposalwould be analogous to the yeast system where change in expression of anexchanged allele (of MAT) leads to induction of downstream cell fatedeterminants that are asymmetrically apportioned to daughter cells. Onlyin the yeast case, an allele is transferred to an expressiblechromosomal site (so that it can be expressed) whereas in the proposedhuman case, the reverse occurs but with the same outcome, i.e. anexpressible chromosomal configuration is transferred to the allele (sothat it can be expressed).

By this hypothesized model, therapy devised to activate this pathway(e.g., at the level of allelic pairing/exchange) would force a(symmetrically-dividing) cancer stem line cell to undergo asymmetricmitosis. In the opposite manner, interruption of allelicpairing/exchange in a cancer stem line would freeze a cancer stem linein a symmetric mitotic mode—a result which could, paradoxically, also bebeneficial for cancer treatment, as will be discussed in subsection 3).

The following are examples of functions and gene products that may actas “bottlenecks” in the asymmetric mitotic pathway involving allelicpairing/exchange:

-   -   a) chromosomal pairing/unpairing—Helicases: Bloom's syndrome        (BS), Werner's syndrome (WS), xeroderma pigementosa, Cockayne's        syndrome, trichothiodystrophy), ATM, topoisomerase-2. Meiotic        pairing genes: multiple yeast genes, as well as human, e.g.,        mos, MLH-1, PMS-2, BRCA-1, SIR4(yeast), RAD50(yeast)    -   b) epigenetic pairing/exchange-zeste (Drosophila), and other        trans-sensing factors.        -   15q11-13 imprinted genes: ZNF127, IPW, PAR5, PAR1, SNRPN,            E6-AP ubiquitin-protein ligase UBE3A).        -   11p15 imprinted genes: IGF-2, IGF-2r, H19, p57KIP2    -   c) downstream of chromosomal pairing—polycombgenes (Drosophila),        transcription factors (e.g., WT-1), APC    -   d) modifiers of epigenetic exchange —        -   X-linked modifier of HD trans-sensing:        -   M31/HSM1, M32    -   e) effectors of epigenetic exchange—        -   alternate splicing (by RNA-binding proteins) of a host of            factors in Drosophila, e.g.: Sxl, dsx    -   f) resolution of recombination intermediates (e.g.,        heteroduplexes, triplexes)—multiple yeast recombination and        repair proteins, endonucleases    -   g) abortion of recombination—mismatch repair, methylation        maintenance proteins, methylation or nick strand recognition        factors        (Dittrich et al, “Imprint switching on human chromosome 15 may        involve alternate transcripts of the SNRPN gene”, Nature Genet.,        14:163-170 (1996); Singh et al, “A sequence motif found in a        Drosophila heterochromatin protein is conserved in animals and        plants”, Nucleic Acids Res., 19:789-794 (1991); Laird, “Proposed        genetic basis of Huntington's disease”, Trends Genet., 6:242-247        (1990); MacDougall et al, “The developmental consequences of        alternate splicing in sex determination and differentiation in        Drosophila”, Dev. Biol., 172:353-376 (1995)).

Accordingly, such processes (and those factors involved) may be added tothe list of effectors of the machinery of asymmetric stem cell mitosisidentified in Table 1. As will be discussed in this section, it followsthat therapeutic activation, in a (symmetrically-dividing) cancer stemline, of native factors that drive asymmetric mitosis of normal stemcells (e.g., but not exclusive to, those mentioned in Table 1 and supra)should induce a cancer stem line to switch to asymmetric mitoticdivision.

As described, by the OSES model symmetric stem cell mitosis (i.e.cancer) initiates when local differentiation-inducing signals within astem cell milieu are disturbed (e.g., due to carcinogen-induceddisruption of those non-stem cells within a stem cell milieu thatnormally produce such differentiation-inducing factors). Accordingly,when such signals are disturbed. 1) differentiation-related genes arenot turned on in stem cells, and 2) underlying structural asymmetry of astem cell becomes “ignored” (i.e., native inhibition of asymmetricmitosis-inducing factors is maintained rather than inhibited) therebypreventing asymmetric segregation of cell fate-determining factors toresulting daughter cells. Accordingly, such a stem cell (i.e., within adisturbed milieu) would undergo mitosis without induction ofdifferentiation-related genes or its asymmetric machinery, which bydefinition is a symmetric mitotic division of stem cellfate-determinants (i.e., the initiation of cancer). By this model, novelOSES-derived therapies designed to activate (i.e., cause to “recognize”)asymmetric mitosis-inducing factors within a cancer stem line shouldlead to coercion of a cancer stem line to divide via asymmetric mitosisthereby leading to its containment. Evidence that cancer cells canindeed maintain an ability to differentiate (i.e., undergo asymmetricmitosis) has been presented in the Background section, and evidence thatcancer cells at the tumor periphery are more differentiated than atcentral regions is consistent with the OSES-derived idea that cancercell differentiation results (not from intrinsic causes but rather) fromlocal differentiation-inducing signals, as described. This OSESscenario, as described, depicts a tumor with a propensity for its most“shielded” cells (i.e. centrally-located ones) to be leastdifferentiated (i.e. the cancer stem line).

It should be emphasized that it is the OSES model (more so thanconventional ones which themselves view the relation of cancer to tissuedevelopment simply as a stochastic result of dedifferentiation), whichseeks to exploit the rapidly advancing fund of knowledge concerning geneproducts involved in normal tissue development (e.g., but not exclusiveto those mentioned in Table 1) for potential therapeutic benefit.Conventional models, in contradistinction, have viewed cancer cellslargely as “alien-like” (e.g., proliferative invasive mutants) and thushave not been as concerned (as the OSES model is) with the parallelsbetween cancer and normal tissue development.

As will be discussed, design of novel OSES-derived therapies will bebased on knowledge of those native factors which normally act in theasymmetric mitotic pathway in stem cells (Table 1) for a preliminarylist) so that such factors can be activated/inhibited, as the case maybe, for the purpose of coercing a cancer stem line to undergo asymmetricmitosis. The pace of current advances in developmental biology is suchthat this list (Table 1) continues to grow and the underlying hierarchyof action regularly updated. Elucidation of the evolutionarily conservedpathway of tissue development (i.e., asymmetric mitosis) in non-humanorganisms will undoubtedly assist in understanding normal human tissuedevelopment (and human cancers). Moreover, following identification ofnon-human development-related genes, more human homologs of these genescan then be isolated by methods used by those skilled in thewell-described art of gene cloning, e.g., via low stringencyhybridization in Southern blotting and/or (preceded by) use ofdegenerate primers to conserved regions of such sequences in PolymeraseChain Reactions (for the purpose of cloning preliminary genefragments)—the actual human genes or sequences of which can then be usedas effectors or templates, respectively, for construction of noveltherapies in the manners to be discusses.

Novel OSES-derived cancer treatments which attempt to switch cancer stemline mitosis from symmetric to asymmetric (thereby altering tumor growthkinetics for the better) include certain methods designed to activatethe native asymmetric mitotic program inherent in all stem cells (andthus in all cancer stem line cells). This may be accomplished at thelevel of: i) ligand-receptor binding, or ii) beyond (e.g., signaltransduction, transcription activation/inhibition, “bottlenecks” such assomatic allelic pairing, or unequal distribution of factors to daughtercells) (see Table 1), with special attention to iii) comprehensivecombination therapy utilizing multiple points of intervention in theasymmetric mitotic pathway, possibly but not necessarily used incoordination with standard chemotherapies so as to simultaneously targetboth the cancer stem line as well as its progeny.

As will be discussed, such interventions can be in the form of deliveryto a cancer stem line of native factors (e.g., ligands, receptors,downstream intracellular elements) that normally drive asymmetric stemcell mitosis or, alternatively, delivery of artificially constructedfactors designed to block native factors that normally inhibitasymmetric stem cell mitosis. As will be discussed, both of thesetherapies upon introduction to a cancer stem line should result inactivation of its asymmetric mitotic pathway.

In addition, an alternative OSES-derived approach to cancer therapy willbe presented in subsection 3) whereby a cancer stem line (rather than beforced to undergo asymmetric mitosis) is paradoxically frozen in asymmetric mitotic program but concomitantly forced to differentiate (inother words, the normally tightly linked processes of differentiationand asymmetric mitosis are uncoupled)—a novel therapy which attempts tocompletely eradicate a cancer stem line, as will be discussed.

i) Activation of Asymmetric Cancer Stem Line Mitosis at theLigand-Receptor Level

Section (I.) describes the use of known differentiation-inducing agents(e.g., ATRA) but in a novel (OSES-derived) dosing manner for the purposeof inducing asymmetric cancer stem line mitosis in cancer treatment,e.g., at differing dosages and dosing intervals. This section (II.)concerns the design of new more specific is differentiation-inducingtherapies for cancer, based on knowledge of the native pathway ofasymmetric stem cell mitosis.

As mentioned, the pathway to asymmetric stem cell mitosis is initiatedby locally-acting differentiation-inducing factors which bind stem cellreceptors—an action that in turn leads to signal transduction resultingultimately in asymmetric mitotic stem cell division (Table 1, FIG. 4).Accordingly, it follows from these ideas that one novel method of cancertherapy would be to deliver such native locally-actingdifferentiation-inducing factors (i.e., those ligands that normallyinduce stem cells to undergo asymmetric mitosis) to a cancer stem linefor the purpose of coercing a switch from symmetric to asymmetric cancerstem line mitosis. This would occur since a cancer stem line shouldexpresses certain cell-surface receptors (having inherited them en blocfrom normal stem cells) that lead to transduction of signals resultingin asymmetric mitosis.

Such native differentiation-inducing factors to be delivered to a cancerstem line would include, but not be exclusive to, gene products of Wnt,Wnt inhibitors, Hedgehog, Transforming growth factor, Epidermal growthfactor among others (some of the receptors of which are listed insection (a) of Table 1). Identification of additional ligands which actin normal tissue development in a stem cell milieu will serve to add tothis list of potential therapeutic effectors. Specifically, suchfactors, if properly delivered to a cancer stem line, will function ashighly-specific therapeutic effectors of asymmetric cancer stem linemitosis (in an analogous fashion to their native role as inducers ofasymmetric mitosis in normal stem cells). Such factors could be injecteddirectly into a tumor, or given intravenously (as is done with ATRA).Additional modifications to such ligands may be necessary to promotetheir stability for use as drugs—a process familiar to those skilled inthe art of drug design and delivery.

Another method of activating, in a cancer stem line, the ligand-receptorportion of the asymmetric mitotic pathway would be via delivery, to acancer stem line, not of ligand but of its cognate receptor (i.e. onewhich normally transduces signals that cause stem cells to undergoasymmetric mitosis) (section (a) of Table 1). This method may be moreadvantageous than delivering ligands in that it may more. “permanently”effect asymmetric cancer stem line mitosis without the requirement forprolonged therapy (as would be necessary when deliveringdifferentiation-inducing ligands whether native or otherwise whosetransduction effects are transient). In other words, targeted deliveryof factors which cause an irreversible switch in the cancer stem line topermanent (rather than transient) asymmetric mitosis would be preferred.This would involve for example, but not be exclusive to, delivery to acancer stem line of a constitutively active receptor (e.g., viagene-directed therapy) which induces downstream events that normallylead to asymmetric mitosis in stem cells.

For example, by the OSES model, delivery to a cancer stem line of aconstitutively active receptor which normally functions in transductionof signals leading to asymmetric stem cell mitosis should result in aswitch to asymmetric cancer stem line mitosis. In general, in vitrodelivery (via infection or transfection) of genes encoding cellreceptors can be used to activate pathways downstream of such receptorsin host cells—a method which has been previously and extensivelydescribed in other circumstances. By the OSES model, infectionspecifically of a cancer stem line, in vivo, would require utilizationof current gene therapy techniques—a technology which continues to beperfected by those skilled in the art. At present, gene-directedtherapies have used both viral (e.g., adenovirus, adeno-related virus,retrovirus) as well as non-viral means of delivery (e.g., naked DNA,chromosomal, or liposomal) (Calos, “The potential of extra chromosomalreplicating vectors for gene therapy”, Trends Genet., 12:463-466(1996)). Accordingly, an OSES-derived gene therapy would involvedelivery of a receptor-encoding gene (as part of either a viral ornon-viral construct) into a cancer stein line either via injection intoa tumor or possibly intravenously. Following administration of such adrug, infection of a cancer stem line with the constitutively activereceptor would be expected to lead to constitutive switch from symmetricto asymmetric mitosis in the cancer stem line thereby permanentlyaltering tumor growth kinetics. Possible receptors to use for such novelOSES-derived therapy include, but are not exclusive to, the mammalianhomologs of Drosophila frizzled (i.e., Wnt receptor) and smoothened(i.e., Hedgehog receptor), PTC, as well as receptors for Transformingand Epidermal growth factors (Perrimon, “Serpentine Proteins Slitherinto the Wingless and Hedgehog Fields”, Cell, 86:513-516 (1996)).Identification of other receptors which normally act in induction ofasymmetric stem cell mitosis (e.g., via investigation of normal tissuedevelopment) will add to the list (section (a) of Table 1) of possibletherapeutic effectors of asymmetric cancer stem line mitosis in themanner described.

It should be noted that these OSES-derived therapeutic goals differsignificantly from those espoused by current cancer gene therapists whohave sought to destroy or correct mutant-cells (i.e., rather than forcea relatively mutationally-spared cancer stem line to regress, asdirected by the OSES model).

Certain technological difficulties associated with gene therapy ingeneral are shared by both current gene therapists as well as by thenovel OSES-based methods proposed here. For one, improved targeting ofgene therapy to stem cells has been a goal for current gene therapistsand likewise would be also be the case for OSES-based therapies.Targeting stem cells is important for classical uses of gene therapybecause it permits permanent gene introduction (i.e. into an immortalcell type) without the need for multiple treatments (which is associatedwith certain complications such as tolerance by virtue of acquiredimmunity to the viral vector). OSES-derived methods would also benefitfrom targeting stem cells as it would similarly limit the number ofnecessary treatments as well as potential toxicities to non-stem cells.Such technological advances in stem cell targeting may be assisted byfurther study of stem cell expression profiles (e.g., following stemcell cloning and expression analysis) in order to identify stemcell-specific factors to target. In addition, differential viralinfection of stem cells versus non-stem cells may also provideinformation on certain stem cell-specific targets to exploit fortherapy. For example, polyomavirus, papillomavirus, parvovirus,Epstein-Barr virus, and cytomegalovirus all display elevated viral geneexpression and gene amplification in differentiating cells while onlylow level episomal DNA maintenance in stem cells (Villarreal,“Relationship of Eukaryotic DNA Replication to Committed GeneExpression: General Theory for; Gene Control”, Microbiol. Rev., 512-542(1991)). Elucidation of those intracellular host factors involved inepisomal maintenance might provide potential targets for stem cell (andthus cancer stem line) identification and therapeutic delivery.

It should be noted, however, that OSES-derived therapies require theadditional ability to be able to distinguish normal stem cells fromsymmetrically-dividing ones (i.e., cancer stem line). This may bedifficult since the two likely express many of the same genes and thusmay not be readily distinguishable (other than by their differences ingrowth kinetics). In other words, it is conceivable that both stem cellsand a cancer stem line share many of the same targets and thus wouldboth be concomitantly infected by a given (viral or non-viral) vectorfor gene therapy. Determination of the differences in gene productssegregated in asymmetric versus symmetric mitosis may provide a feasiblemeans to distinguish normal stem cells from a cancer stem line—and thusto exploit for therapeutic gene delivery (to be discussed in more detailin the section (III.). One may also glean expression differences betweennormal stem cells and a cancer stem line for which to exploit fortherapeutic purposes by cloning these cell populations and performingexpression analyses (e.g., via subtraction hybridization, awell-described technique to determine expression differences betweentissues). It is also conceivable that toxicity associated with this typeof novel OSES-based therapy (i.e., induction of asymmetric cancer stemline mitosis) may not be very significant for several reasons. Firstly,one of the major reasons classic gene therapy has encountereddifficulties in infecting normal stem cells is because of architecturalconstraints (i.e., stem cells are sequestered within environmentalniches)—a condition, which ironically might actually be advantageous forOSES-based therapy. More specifically, OSES-derived gene therapy(targeted to both normal stem cells as well as a cancer stem line) mightpreferentially target a cancer stem line because the disorganizedarchitecture of a tumor may actually act as a less significant barrierthan a normal well-defined stem cell niche. In this way, the technicaldifficulties encountered by classical gene therapies could be exploitedby OSES-based therapies. There is an additional reason why the toxicityto normal stem cells associated with this type of therapy (i.e.,induction of asymmetric cancer stem line mitosis) may not be verysignificant. Namely, considering that normal stem cells are presumablyalready dividing via asymmetric mitosis anyway, therapy designed toinduce asymmetric mitosis would not be expected to change theirbehavior—thus no toxicity. However, other OSES-based therapies (that donot simply induce-asymmetric mitosis), to be discussed in subsection 3),may be more toxic to normal stem cells than the therapy described inthis section and thus warrant more specific delivery to the cancer stemline (see Section III.).

ii) Activation of Elements Downstream of Ligand Receptor Interactions toInduce Asymmetric Cancer Stem Line Mitosis

As mentioned, another OSES-derived method of inducing a cancer stem lineto undergo asymmetric mitosis would involve activation (in a cancer stemline) of those elements downstream of ligand-receptor binding thatnormally play a role in signal transduction leading ultimately toasymmetric mitotic division of stem cells. A number of genes and geneproducts involved in asymmetric mitosis have been identified (see Table1, Table 2, Table 3 and Section II, subsection 2)) and may providepotential targets for therapeutic manipulation. More specifically,activation of factors which promote asymmetric mitosis or,alternatively, inhibition of factors which drive symmetric mitosis areeach methods which can be used to effect reversion of a cancer stem linefrom exponential to arithmetic growth. One method by which this could beaccomplished is via delivery to a cancer stem line of a gene itself thatnormally activates the asymmetric pathway or inhibits the symmetricmitotic pathway (downstream of ligand-receptor binding) in adult stemcells (specific examples will be given in the next subsection, iii). Anon-exhaustive list of such gene products is contained in Table 1, Table2 and Table 3, as well as Section II, subsection 2) of this application.Identification of additional products involved in the asymmetric mitoticprogram downstream of receptor binding (e.g., likely to be gleaned fromongoing investigation of normal development) will add to this list ofpotential effectors of OSES-derived cancer therapy. Delivery of suchgenes would involve methods of gene therapy (e.g., utilization of viralor non-viral vectors) via injection or intravenous use in a similarmanner as to that described in the previous subsection (i, for genesencoding receptors). Perfection of such techniques to maximize deliveryto a specific cell population (in this case a cancer stem line) whileminimizing side effects is a major priority, as mentioned before, forboth classic as well as OSES-derived methods of gene therapy.

Again, to reiterate, these OSES-derived therapeutic goals differsignificantly from those espoused by current cancer gene therapists whohave sought to destroy or correct mutant cells (i.e. rather than force arelatively mutationally-spared cancer stem line to regress, as directedby the OSES model).

Knowledge of downstream effectors of asymmetric mitosis can also beexploited for an alternative OSES-derived method of cancer therapy.Namely, in addition to using positively-acting factors themselves (i.e.,those factors that drive asymmetric mitosis) as vehicles of gene therapyfor cancer, knowledge of the DNA coding sequences of those factors thatnormally drive symmetric mitosis (or inhibit asymmetric mitosis) canalso be used as templates for the construction of elements that willblock their inhibitory function (i.e. thereby also leading to asymmetricmitosis of a cancer stem line). For example, such methods would includebut not be exclusive to, therapies that utilize RNA-based antisense orribozyme modalities or other newer technologies which may arise thatallow design of factors that specifically block the function of certaintargeted gene products. Both of these mentioned methods take advantageof sequence information (of a targeted nucleic acid) in order toconstruct a highly specific therapeutic agent which will bind to itscognate (i.e., target) messenger RNA species and inactivate it before itcan be properly translated into functional protein. In this manner, aspecific gene product (e.g., one that normally inhibits asymmetricmitosis) can be blocked. It should be noted that there have been anumber of recent advances in the technical aspects of these therapies(Ellington et al, “Ribozymes in Wonderland”, Science, 276:546547 (1997);Roush, “Antisense aims for a renaissance”, Science, 276:1192-1193(1997)), which should more readily allow for their exploitation fornovel OSES derived uses. Antisense and ribozyme therapies have alreadybeen shown to be efficacious in preliminary cell culture experiments butcompelling evidence for any clinical utility remains to be demonstrated.Others have indeed proposed ribozyme therapy for the treatment of cancer(e.g., via viral or non-viral delivery systems) and have used itsuccessfully in preliminary in vitro experiments (Kashani-Sabet et al,“Suppression of the Neoplastic Phenotype in Vivo by an Anti-rasRibozyme”, Cancer Res., 54:900-902 (1994)). However, it should beemphasized that such experiments were performed and viewed with theconventional model of cancer in mind whereby highly proliferative mutantcells were targeted—which according to the OSES model, is the wrong cellpopulation to target and thus such therapy will leave a cancer stem lineintact. Thus, it is proposed here that such therapies or related-onesshould be used for novel purposes whereby a slow-growing relativelymutationally-spared cancer stem line is targeted.

In addition, in vitro evolution offers another method of perfecting thespecificity of ribozymes, a method which can also be used to targetnon-nucleic acid (e.g., non-messenger RNA) species, e.g., proteins(involved in asymmetric mitosis) (Beaudry and Joyce, “Directed evolutionof an RNA enzyme”, Science, 257:635-641 (1992)). Moreover, newertechnologies may soon improve on the specificity of action of antisenseand ribozyme RNA-based therapies. As mentioned, by the OSES model, suchtherapies should be geared toward targeting the relativelymutationally-spared cancer stem line as described above. Like deliveringgenes themselves (i.e., those that activate asymmetric mitosisdownstream of ligand-receptor binding) to a cancer stem line, deliveryof specifically constructed inhibitors (e.g., but not exclusive to,antisense or ribozyme agents) to a cancer stem line can be performed viaviral or non-viral means, as described by those skilled in the art, forthe purpose of blocking specific factors that normally either inhibitasymmetric mitosis or activate symmetric mitosis thereby leading to thedesired therapeutic effect of asymmetric cancer stem line mitosis.Specific examples will be discussed in the following subsection, (iii).

As discussed in the previous subsection (i), targeting of stem cells,and in particular symmetrically-dividing ones (i.e. the cancer stemline) should be the therapeutic goal.

iii) Combination Therapy to Force Asymmetric Cancer Stem Line Mitosis

Considering the complexity of the asymmetric mitotic pathway (Table 1,Table 2, and Table 3), it is likely that activation of such a pathway ina cancer stem line will require multiple points of intervention. Withthis in mind, a combination of gene-delivered therapy and geneproduct-inhibiting therapy (e.g., antisense, ribozyme) may be necessaryto induce a cancer stem line to assume arithmetic growth kinetics.

Of course, attempts to intervene at “bottlenecks” in this pathway willbe advantageous as they will necessitate fewer therapeutic maneuvers.For example, delivery to a cancer stem line of 1) factors that promoteallelic pairing/exchange (e.g., but not exclusive to, zeste, SNRPN, andothers) via gene therapy—by methods previously discussed, or 2)template-constructed (e.g., but not exclusive to, antisense or ribozyme)inhibitors of factors which block allelic pairing/exchange (e.g., butnot exclusive to zeste-inhibitors, SNRPN-inhibitors) by methodspreviously discussed may alone or in combination lead to a switch fromsymmetric to asymmetric cancer stem line mitosis. It should be notedthat those factors (mentioned in the previous sentence) preliminarilydeemed to promote allelic pairing/exchange may actually block allelicpairing/exchange (and vice versa). In other words, the mechanisms ofallelic pairing/exchange await further clarification, and thus thesementioned examples of therapy may need to be reversed.

While the mechanisms of gene products specifically involved in allelicpairing/exchange await flier description, the mechanisms of action ofother gene products involved in the general (i.e., non-“bottleneck”portion) of the asymmetric mitotic pathway are now beginning to emerge.One such pathway which leads to asymmetric mitosis, namely the Wntsignaling pathway, has begun to emerge from pooled data derived from anumber of different species. Wnt gene products are involved in normalembryonic and adult tissue development (Nusse and Varmus, “Wnt genes”,Cell 69:1073-1087 (1992)). One mechanism of action of these factors isto mark cell polarity and/or permit inherent cell polarity—a maneuverthat leads ultimately to asymmetric mitotic divisions (Herman et al,“The Caenorhabditis elegans gene lin-44 controls the polarity ofasymmetric cell divisions”, Development, 120:1035-1047 (1994)). Asignaling pathway by which this process occurs is now becoming clearer:locally-acting Wnt proteins bind to their receptor (frizzled, inDrosophila) leading eventually to activation of transcription factors(e.g., LEF-1/XTCF-3 in Xenopus) and then to activation of certain cellfate-determining genes (e.g., engrailed, in Drosophila) some of whichthemselves, or downstream products thereof, are unequally segregated todaughter cells. Within this signaling pathway are numerous intermediatepositively-acting factors such as APC and beta-catenin (mammalian), aswell as certainly negatively-acting ones such as GSK-3-beta (Xenopus)—aninhibitor of beta-catenin (Kuhl et al, “Wnt signalling goes nuclear”,Bioessays, 19:101-104 (1997)). It is proposed here that the binding ofcertain Wnt proteins to their receptors (on stem cells of adult tissues)leads to activation of the mentioned pathway and asymmetric stem cellmitosis. By this model, symmetrically-dividing stem cells (i.e., acancer stem line) would maintain inhibition of the Wnt pathway but wouldbe susceptible to having it therapeutically up-regulated (which wouldlead to a switch from symmetric to asymmetric cancer stem line mitosis).

Accordingly, for the purpose of treating cancer via novel OSES-derivedtherapy, one is left with multiple possible sites in the Wnt pathway atwhich to intervene. For example, in order to force asymmetrically-dividing cancer stem line to divide asymmetrically, onecould activate positively-acting factors in the Wnt pathway (e.g., butnot exclusive to APC, beta-catenin, LEF-1/XTCF-3) via gene therapy, i.e.delivery of the coding sequences of such factors to a cancer stem linein a non-viral or viral construct, as discussed in the last 2subsections (i, ii). Alternatively, one may attempt to block thosefactors which normally inhibit the native Wnt pathway (e.g., but notexclusive to, GSK-3-beta), e.g., by designing a “tailor-made” antisenseor ribozyme agent specific for the GSK-3-beta mRNA species anddelivering such a construct to a cancer stem line by methods discussedin the last subsection (ii). By blocking this gene product (i.e. a geneproduct that presumably inhibits asymmetric mitosis), a cancer stem linewould be forced to assume asymmetric mitotic division. Identification ofother factors in the Wnt pathway will add to the list of potential genesto use in gene therapy as activators of asymmetric cancer stem linemitosis, or as templates for the construction of (e.g., but notexclusive to, antisense or ribozyme) inhibitors to block symmetricmitosis.

In addition to the Wnt pathway, there are also cumulative data emergingconcerning other asymmetric mitosis-related pathways. For example, thereis evidence that one highly conserved factor (cdc42) involved in yeaststructural asymmetry has a downstream kinase target in humans. Such acandidate kinase (Kp78) in humans appears to be related to a gene(par-1) in nematodes which is known to affect the asymmetric action ofanother nematode factor (SKN-1) which itself is related to the yeastHO-inhibiting factor (Ash-1p). Thus, assuming the likely existence of aconserved human homolog for each of these genes, a potential pathwayemerges whereby structural asymmetry mediated by cdc42 leads to theasymmetric action of a human kinase (Kp78) which activates a humanSKN-1-homolog that in turn activates a transcription factor (e.g., humanAsh-1p-related homolog) that effects expression of cell fate-determininggenes (e.g., human HO-related gene product, possibly also like in yeastis involved in chromosome pairing/strand exchange). The involvement ofsuch a gene in pairing/-exchange suggests it may act at a key“bottleneck” in the asymmetric mitotic pathway (FIG. 4). Accordingly,OSES-derived therapy might be constructed to activate positively-actingfactors (e.g., cdc42, Kp78/par-1, SKN-1, HO), or block negatively-actingfactors (e.g. Ash-1p) by the methods of gene therapy and gene-inhibitor(e.g., antisense and ribozyme) therapy described in this and previoussubsections. It should also be noted that those factors (mentioned inthe previous sentence) deemed to be positively-acting may actually benegatively-acting in asymmetric mitosis, and vice versa, and were placedin these categories here only as a preliminary gesture and are notintended to be final categorizations of gene function. Such finalcategorizations await additional evidence from investigationalscientists working in these areas of developmental biology.

Interestingly, human Kp78, which as mentioned is related to nematodepar-1, has been found to be deleted in certain human cancers—a findingconsistent with the OSES model and not necessarily predicted or expectedby conventional models (Way et al, “Cell polarity and the mechanism ofasymmetric cell division”, Bioessays, 16:925-931 (1994); Lin et al,“Neuroblasts: a model for the asymmetric division of stem cells”, TrendsGenet., 13:33-39 (1997)). In other words, by the OSES model deletion ofan asymmetric mitosis-related gene (e.g., Kp78) might be expected to befound among a sub-population of peripherally-located progeny of a cancerstem line, i.e., where selection for differentiation-defectivenessoccurs. Conventional models, however, predict deletions to occur mostlyin classic tumor suppressor genes (e.g., RB-1, TP53, BRCA-1) and thusKp78 deletion would be unexpected.

The OSES model would predict that other genes involved in normal tissuedevelopment (and more specifically in the induction of asymmetric stemcell mitosis) should also be found to be altered in progeny of thecancer stem line. Interestingly, there is evidence that PTC (patchedhomolog) is mutated in human basal cell carcinomas, Wnt genes areinvolved in mammary tumorigenesis, and alteration of hedgehog expressionmay contribute to neoplasia (Johnson et al, “Human Homolog of patched, aCandidate Gene for the Basal Cell Nevus Syndrome”, Science,272:1668-1671 (1996); Tsukamoto et al, “Expression of the int-1 gene intransgenic mice is associated with mammary gland hyperplasia andadenocarcinomas in male and female mice”, Cell, 55:619-625 (1988)).

As previously mentioned, there is increasing evidence that gene productsinvolved in the asymmetric mitotic pathway are highly conserved in bothstructure and function among distant species (e.g., cdc42 in yeast,nematode, and human) (Way et al, “Cell polarity and the mechanism ofasymmetric cell division”, Bioessays, 16:925-931 (1994); Lin et al,“Neuroblasts: a model for the asymmetric division of stem cells”, TrendsGenet., 13:33-39 (1997)). Accordingly, improved description of theasymmetric mitotic pathway in cells of non-human organisms will likelyprovide insight into the same process in human cells. Likewise,identification of additional gene products involved in these processesin any of a number of different organisms will not only help describethe process of asymmetric mitosis but will also provide additionalmolecular probes as well as key sequence information to use for thepurpose of cloning their human homologs. In addition to some of theabove mentioned pathways of asymmetric mitotic division, there arecertainly others to be more well-described in the near future. Some ofthese may include the hedgehog, patched, transforming and epidermalgrowth factors pathways (Table 1). However, this is certainly notintended to be an exhaustive list.

It is expected, based on the teachings in this application, that oneskilled in the art will be able to select other suitable targets, e.g.,those mentioned above as well as other gene products (identified in thefuture likely via, but not exclusive to, study of developmental and stemcell biology) for which to construct OSES-based cancers therapies. Morespecifically, knowledge of gene products involved in asymmetric mitosisin human cells will in turn, as directed by the OSES model, provideadditional targets within a cancer stem line for which totherapeutically enact a permanent switch from symmetric to asymmetricmitosis. As mentioned, identification of such mitosis-related genes willallow their use in, but not exclusive to, gene therapy, as well asprovide sequence information necessary for the construction of“tailor-made” inhibitors (e.g., antisense or ribozyme therapies).

As discussed in the previous subsection (i), targeting of stem cells,and in is particular symmetrically-dividing ones (i.e. the cancer stemline) should be the therapeutic goal.

It should be well-noted that this is not intended to be an exclusivedescription of all possible methods which could be derived from the OSESmodel to effect asymmetric cancer stem line mitosis, but rather to serveas examples of some of a number of possible ways in which such novelcancer therapies could be reduced to practice. As alluded to earlier,OSES-based therapies could be used alone or in combination with standardchemotherapies as a 2-pronged attack to both contain the cancer stemline as well as concomitantly lower the tumor burden, respectively.

EXAMPLE 2

A patient is found to have pancreatic cancer by conventional detectiontechniques (e.g., CT scan and biopsy). A ribozyme is constructed thatspecifically blocks a factor (e.g., GSK-3-beta) that normally inhibitsasymmetric mitosis in pancreatic epithelial stem cells. The ribozyme isplaced within a viral vector and delivered intravenously to the patient.This therapeutic vector will force stem cells of pancreatic origin todivide asymmetrically thus causing the cancer stem line fueling thepancreatic tumor to assume arithmetic growth kinetics (see FIG. 3B)without significant toxicity to normal pancreatic epithelial stem cellsthat are dividing asymmetrically anyway.

3) Induction Of Irreversible Stem Line Differentiation

As mentioned, one novel OSES-derived method of treating cancer (asdescribed in subsection 2) is to force a cancer stem line to switch fromsymmetric to asymmetric mitotic division. However, in designing such atherapy, it should be remembered that when a cancer stem line is inducedto differentiate it does so in an asymmetric fashion thereby preservingan immortal stem line which does not itself differentiate (i.e., onedaughter cell remains as a cancer cell thereby maintaining the cancerstem line, while one daughter cell differentiates) (FIGS. 3A and 3B).Accordingly, an alternative OSES-based novel therapy for cancer would begeared toward total eradication of the cancer stem line. Such a therapymight include, but not be exclusive to, switching a cancer stem linefrom a symmetric program of proliferation (not to an asymmetric mitoticprogram but rather) to a symmetric program of differentiation wherebyboth daughter cells of a cancer stem cell are forced to terminallydifferentiate thereby potentially resulting in a complete eradication ofthe stem line and cancer cure (FIGS. 3A and 3B).

In order to induce a cancer stem line to undergo symmetricdifferentiation, one may employ methods such as but not exclusive to: 1)paradoxical inhibition of the asymmetric mitotic machinery therebyeffectively “freezing” a cancer stem line in a symmetric mitotic mode,followed by ii) induction of cancer stem line differentiation which mustthen proceed symmetrically. This technique would effectively uncouplethe normally tightly-linked processes of asymmetric mitosis anddifferentiation. It should be noted that asymmetric mitosis anddifferentiation, while tightly-linked processes in mammals, are oftenindependent processes in lower eukaryotes (Horvitz et al, “Mechanisms ofAsymmetric Cell Division: Two Bs or Not Two Bs, That is the Question”,Cell, 68: 237-255 (1992); Villarreal, “Relationship of eukaryotic DNAreplication to committed gene expression: general theory for genecontrol”, Microbiol. Rev., 512-542 (1991)).

i) Paradoxical Inhibition of Asymmetric Cancer Stem Line Mitosis

One means of blocking asymmetric mitosis of a cancer stem line is thereverse of that mentioned in the previous subsection 2), i.e. blockrather than activate those factors which drive asymmetric mitosis and/oractivate rather than block those factors inhibit asymmetric mitosis(Table 1). This may be accomplished in a similar manner as thatmentioned in the previous section, i.e. via gene-delivered therapy orgene product-inhibiting therapy (e.g., antisense, ribozyme) to a cancerstem line but with the opposite desired effect. For example, rather thandeliver to a cancer stem line a receptor that normally activatesasymmetric mitosis (e.g., but not exclusive to, fried), one shoulddeliver an inhibitor of this pathway (e.g., but not exclusive toGSK-3-beta or Ash-1p). Alternatively, one could construct an“tailor-made” inhibitor (e.g., antisense or ribozyme) to apositively-acting factor (e.g., but not exclusive to, beta-catenin), ora “bottleneck” element (e.g., but not exclusive to, elements thatpromote allelic paring/exchange such as SNRPN) in the asymmetric mitoticpathway. It should be noted that blocking the asymmetric mitoticmachinery of a cancer stem line is a seemingly risky approach because itwill effectively “freeze” the cancer stem line, paradoxically, in anexponential growth phase. This method of therapy has the opposite goalto that proposed in the previous subsection 2) which seeks to switch acancer stem line to an asymmetric mitotic mode. Accordingly, by thisalternative method, a cancer stem line “frozen” in a symmetric mitoticmode, if left unchecked, would continue to proliferate exponentially.Thus, this initial intervention must be promptly followed by inductionof cancer stem line differentiation—which would then proceed without theasymmetric machinery (i.e., would proceed in a symmetric fashion sincethe asymmetric machinery has been blocked). Accordingly, cell fate(i.e., differentiation-determining) elements will be equally segregatedto daughter cells thereby extinguishing the immortal stem line.

ii) Induction of Symmetric Cancer Stem Line Differentiation

Following a block to asymmetric cancer stem line mitosis as describedabove in (i) (i.e., “freezing” a cancer stem line in a symmetric mitoticmode), differentiation-induction should then proceed symmetricallyresulting in eradication of the cancer stem line. By this method, thenormally tightly linked processes of asymmetric mitosis anddifferentiation become uncoupled.

There are a number of possible methods by which to induce cancer stemline differentiation. For example, as mentioned in the previoussubsection 2), delivery to a cancer stem line ofdifferentiation-inducing agents (e.g., but not exclusive to, ATRA),differentiation-inducing ligands (e.g., but not exclusive to Wnt, seeTable 1) or receptors in the asymmetric mitotic pathway (e.g., but notexclusive to Sizzled, see Table 1) via the methods described in theprevious section (while proposed to induce asymmetric mitosis when acancer stem line is not “frozen” in a symmetric mitotic mode, as insubsection 2), is proposed here (as a result of uncoupling asymmetricmitosis from differentiation) to induce symmetric differentiation.Activation of positively-acting (or inhibition of negatively-acting)elements in the asymmetric mitotic pathway downstream of ligand-receptorbinding, by the methods described in the previous subsection 2) (e.g.,gene therapy, or antisense/ribozyme therapy), may also be employed inthis scenario to induce differentiation in a cancer stem line. In thiscase, however, it would behoove one to intervene at the more upstreampoints (e.g., prior to “bottlenecks”) which are dominated more bydifferentiation-related processes, rather than at the more dot points(e.g., “bottlenecks” or beyond) which are dominated more by asymmetricmitotic-related processes. In other words, the goal is to inducedifferentiation products so that they may be equally delivered to bothdaughter cells. Thus, it is a hypothesis of the OSES model thatdifferentiation induction of a stem cell normally triggers an asymmetricmitotic mechanism such that a therapeutic block to this trigger willallow differentiation to occur without activation of the asymmetricmachinery (i.e. and thus symmetrically).

There may be additional methods by which to induce a cancer stem line toactivate differentiation-related gene products. For example, it has beenshown that starvation can induce cells to become growth quiescent and attimes differentiate (or under certain conditions undergo apoptosis,i.e., programmed cell death) (Hoffman and Lieberman, “Molecular controlsof apoptosis: differentiation/growth arrest primary response genes,proto-oncogenes, and tumor suppressor genes as positive and negativemodulators”, Oncogene, 9:1807-1812 (1994); Sherley et al, “Expression ofthe wild type p53 antioncogene induces guanine nucleotide-dependent stemcell division kinetics”, Proc. Natl. Acad. Sci. USA, 92:136-140 (1995)).Considering evidence that certain starvation-induced responses (e.g.,growth arrest/differentiation) occur asymmetrically (i.e., withpreservation of a stein line of cells capable of proliferative growthfollowing discontinuation of starvation conditions) (Sherley et al,“Expression of the wild type p53 anti-oncogene induces guaninenucleotide-dependent stem cell division kinetics”, Proc. Natl. Acad.Sci. USA, 92:136-140 (1995)), it is proposed here that starvationconditions may approximate differentiation-induction and thus beemployed for the purposes of novel OSES-based cane therapy. In otherwords a starvation-treated cancer stem line, following prior blockage ofthe asymmetric mitotic machinery of a cancer stem line (by the methodsdescribed in this section), would (like that for adifferentiation-induced cancer stein line “frozen” in a symmetricmitotic mode) also be expected to cause growth arrest/differentiation(or apoptosis) of a cancer stem line in a symmetric manner therebyresulting in its eradication.

A related case may provide some preliminary support for such novelproposals. It has been previously shown that certain starved cells inculture undergo a growth quiescent phase which has been bettercharacterized as an asymmetric mitotic/differentiation-like program(Sherley et al, “Expression of the wild type p53 anti-oncogene inducesguanine nucleotide-dependent stem cell division kinetics”, Proc. Natl.Acad. Sci. USA, 92:136-140 (1995). Interestingly, other cell types inculture, which undergo a similar growth quiescent phase in response tostarvation conditions, undergo apoptosis (presumably an alternate branchof the differentiation pathway) if their mitotic machinery is altered(e.g., by c-myc deregulation) (Evan et al, “Induction of apoptosis infibroblasts by c-myc protein”, Cell, 69:119-2128 (1992); Hermeking andEick, “Mediation of c-Myc-induced apoptosis by p53”, Science,265:2091-2093 (1994)). It is proposed here that this differentiation (inthis case apoptotic) response to starvation by c-myc deregulated cellsis analogous to the hypothesized (symmetric) differentiation response tostarvation by a cancer stem line. “frozen” in a symmetric mitotic mode.In other words, it is proposed here that c-myc deregulation in cells inculture approximates a block to the asymmetric mitotic machinery in acancer stem line, both of which scenarios would respond to starvation byundergoing a terminal process (e.g., apoptosis or symmetricdifferentiation, respectively).

Starvation has been induced by a host of methods including, but notexclusive to alterations in enzymes involved in nucleic acid synthesis(e.g., GTP), removal of essential growth factors, certain drugs andchalones (Hoffman and Lieberman, “Molecular controls of apoptosis:differentiation/growth arrest primary response genes, proto-oncogenes,and tumor suppressor genes as positive and negative modulators”,Oncogene, 9:1807-1812 (1994); Sherley et al, “Expression of the wildtype p53 anti-oncogene induces guanine nucleotide-dependent stem celldivision kinetics”, Proc. Natl. Acad. Sci. USA, 92:136-140 (1995).Accordingly, these as well as other strategies not mentioned here couldbe used to induce cancer stem line starvation. Preliminary testing ofthis method in cultured cells would involve initially “freezing” acancer stem line a symmetric mitotic mode (by methods described in thissection), and then inducing either differentiation (by the methodsdescribed in this section), or starvation (by addition of certain agentsused by investigators to induce starvation, some of which are mentionedin this section). Measurement of growth kinetics of the stem line ofsuch cells in culture, a technique previously described (Sherley et al,“Expression of the wild type p53 anti-oncogene induces guaninenucleotide-dependent stem cell division kinetics”, Proc. Natl. Acad. SciUSA, 92:136-140 (1995)), will determine whether a shift to symmetricdifferentiation/-apoptosis has occurred. Such therapies could then beutilized in genetically-altered mouse models of cancer, using genetherapy vehicles (e.g., viral or non-viral), as mentioned, and then inhumans.

It should be well-noted that this is not intended to be an exclusivedescription of all possible methods which could be derived from the OSESmodel to effect terminal differentiation/apoptosis, but rather to serveas examples of some of a number of possible ways in which such novelcancer therapies could be reduced to practice. As discussed in theprevious subsection (i), targeting of stem cells, and in particularsymmetrically-dividing ones (i.e. the cancer stem line) should be thetherapeutic goal. In addition, as alluded to earlier, OSES-basedtherapies could be used alone or in combination with standardchemotherapies as a 2-pronged attack to both destroy the cancer stemline as well as concomitantly lower the tumor burden, respectively.

EXAMPLE 3

A patient is found to have lung cancer by conventional detectiontechniques (e.g., CT scan and biopsy). A ribozyme is constructed thatspecifically blocks a factor (e.g., SNRPN) that normally activatesasymmetric mitosis in lung epithelial stem cells. The ribozyme is placedwithin a viral vector and delivered intravenously to the patient. Thistherapeutic vector will force stem cells of lung origin to dividesymmetrically thus causing the cancer stem line fueling the lung tumorto assume exponential growth kinetics. This maneuver is quickly followedby intravenous administration of a starvation-inducing agent e.g.,inhibitor of nucleic acid synthesis) which forces the cancer stem lineto initiate its differentiation program but in a symmetric mannerthereby extinguishing the stem line (see FIG. 3C). Toxicity to normalstem cells may be minimal due to their environmental sequestration.Efforts to limit this toxicity further are discussed in the next section(III.).

III. Targeting Symmetrically-Dividing Stem Cells (Cancer Cells) WhileSparing Asymmetrically Dividing (Normal) Stem Cells.

As discussed in section II., (2), end of subsection (i), technologicaladvances in gene therapy will allow OSES-based cancer therapy to bebetter directed toward stem cells (by some of the means discussed) andmore specifically toward the cancer stem line. While toxicities based onmethods discussed in section II., (2) may not be very significant (sincesuch methods would simply force a normal stem cell to undergo its nativemitotic mode anyway, i.e., asymmetric mitosis), toxicities based onmethods discussed in section II., (3) may be more significant since theycould potentially also cause normal stem cells to undergo symmetricdifferentiation/apoptosis (i.e., eradication) thereby leading to aninability of certain tissues to renew themselves. Accordingly, it wouldbe beneficial, at least for this latter method of therapy, to avoidnormal (asymmetrically-dividing) stem cells and just target the(asymmetrically-dividing) cancer stem line.

It is, however, conceivable, as previously mentioned, that because ofarchitectural constraints (i.e. stem cells are sequestered withinenvironmental niches), OSES-derived gene therapy (targeted to bothnormal stem cells as well as a cancer stem line) might preferentiallytarget a cancer stem line because the disorganized architecture of atumor may actually act as a less significant barrier than a normalwell-defined stem cell niche. Experimental verification for such an ideais needed. In the meantime, efforts to distinguish normal stem cellsfrom a cancer stem line for the purpose of therapeutic targeting iswarranted.

There are several ways in which to preferentially targetsymmetrically-dividing stem cells (i.e. a cancer stem line) withOSES-based therapy while sparing normal (asymmetrically-dividing) stemcells. The following are possibilities, but are not intended to becomprehensive, as other possibilities undoubtedly exist or will existwhen new technologies arise.

Firstly, determination of differences in cell surface antigens between acancer stem line and normal stem cells may permit design of vectors(viral or non-viral) which can preferentially bind to those antigenssolely on a cancer stem line. One may glean expression differences (withparticular attention paid to cell surface antigens) between normal stemcells and a cancer stem line by cloning these cell populations andperforming expression analyses (e.g., via subtraction hybridization, awell-described technique to determine expression differences betweentissues), or by protein purification and comparison of respective cellsurface antigens. In addition investigation of the mechanisms by whichcertain viruses differentially infect and/or act in cells of differingmitotic modes may also yield gene products (e.g., cell surface antigens)specific to cells that divide symmetrically versus asymmetrically(Villarreal, “Relationship of Eukaryotic DNA Replication to CommittedGene Expression: General Theory for Gene Control”, Microbiol. Rev.,512-542 (1991)).

Secondly, rather than distinguishing a cancer stem line from normalstein cells for targeted therapy by virtue of differences in cellsurface antigens, one may utilize expression differences in certainintracellular gene products. This would then allow the following noveltechnique: pre-treatment via delivery of a “tailor-made” pre-treatmentfactor (PTF) which: 1) binds an intracellular element maintained solelywithin a cancer stem line (i.e. and not in normal stem cells) and 2) isrequired (e.g., act as a cofactor) for the function of an OSES-basedtherapy (e.g., asymmetric mitosis-inhibiting factor, AMIF to be givenfollowing this pre-treatment. Such a technique will allow non-targeteddelivery of AMIF to both a cancer stem line as well as normal stemcells, but will ensure AMIF activity solely in a cancer stem line, byvirtue of the presence of a “tailor-made” PTF (in a cancer stem line butnot in normal stem cells) which is necessary for AMIF function.Following pre-treatment in this manner and subsequent delivery of theAMIF (which by this method would only be active in the cancer stem lineand not in normal stem cells), induction of differentiation/starvationcan proceed for the purpose of eradicating a cancer stem line whilesparing normal stem cells. For design of a PTF, one must identify asuitable intracellular element that is cancer stem line-specific forwhich to specifically bind. Qualifying as cancer stem line-specificintracellular elements may be notch, numb, or other intracellularelements (see Table 1, Table 2, and Table 3) unequally segregated duringasymmetric mitosis to the non-stem cell daughter (i.e., purged fromnormal stem cells), but which may also be equally segregated to bothdaughter cells during symmetric mitosis (see FIGS. 5A and 5B).Accordingly, design of a PTF to specifically bind such a cancer stemline-specific intracellular element may proceed, e.g., but not exclusiveto, via use of known cofactor binding sites (of that particularintracellular element), or via in vitro evolution selecting forribozymes which specifically bind (but do not cleave) a cancer stemline-specific intracellular element. In addition, the PTF must also bedesigned with a “tailor-made” moiety which as proposed would be required(e.g., act as a cofactor) for AMIF function. Such a moiety could be (orcode for) a cofactor, chaperone, activating enzyme, activating ribozyme,cleavage ribozyme, or other factor which would activate or be necessaryAMIF function. For example, if the AMIF were a ribozyme, then a portionof the PTF might be a ribozyme which cleaves or folds the AMIF atsome-point to make it active (see FIGS. 6A and 6B).

Novel Detection Methods Provided by the Invention

Conventional cancer screening methods include, among others: physicalexam, mammography (breast cancer), PAP smear (cervical cancer), stoolguaiac and colonoscopy (colon cancer), PSA (prostate cancer), skinsuveillance (skin cancer), sputum analysis and chest X-ray (lungcancer), among others. For a positive test result, however, all of thesemethods unfortunately require that a significant number of cancer cellsbe present in the examined patient. Accordingly, such “early” detectionmethods are often too late. For example, approximately 30% of patientswith “early” breast cancer without gross evidence of lymphatic spreadwho have undergone “complete” surgical resection will still developrecurrent disease at a later date. In addition,radiographically-detectable lung tumors are often incurable despitetheir seemingly “small” size. These disturbing findings indicate thatconventional detection techniques do not identify cancer early enough.

By the OSES model, however, cancer cells are produced not by a gradualevolutionary process at the cellular level but rather via a one stepswitch in stem cell mitotic mode. Accordingly, a potentially detectableneoplastic lesion would be expected to be present prior to attaining thelarge size (in numbers of cancer cells) required by conventionaldetection techniques.

Identification of symmetrically-dividing stem cells (i.e. a cancer stemline) provides, according to the OSES model, a novel and extremelysensitive method for the early detection of cancer. In order to do this,one must identify elements which are specific to the cancer stem lineand then design factors which will bind specifically to them—whichthemselves are readily detectable. This may be effected, e.g., byattaching a readily detectable moiety (e.g., a fluorescent tag orradionuclide) to a cancer stem line-specific binding factor. Thistechnique will enable a cancer stem line to be readily detected, e.g.,by infusing such a cancer stem line-specific binding factor (with anattached detectable moiety) into a patient and then placing the patientinto a total body scanner/developer which can sense the attached moiety(e.g., fluorescent tag or radionuclide) and record its presence andbodily location on film (e.g., this could be accomplished by usingfluorescent-sensitive film, or a CT or MRI scanner). In this manner, thedetection of tag (e.g., fluorescence, radionuclide) in aberrant patternswithin a tissue (i.e., different from the patterns of normal stem cellswithin a tissue) would indicate a mass of symmetrically-dividing stemcells thereby detecting early carcinogenesis. Following identificationof symmetrically-dividing stem cells (i.e., a cancer stem line), such alesion could be treated locally with; e.g., with conventional therapysuch as irradiation or surgical excision much earlier than would bepossible by convention detection/treatment methods.

Factors specific to the cancer stem line would include: surfaceantigens, intracellular factors which are differentially apportioneddepending on mitotic mode (e.g., of the type unequally apportionedduring asymmetric mitosis (e.g., but not exclusive to, notch, numb) asdescribed previously in section (III.) (see: FIGS. 5A and 5B).Construction of factors which will specifically hind to these cancerstem line-specific entities include monoclonal antibodies to surfaceantigens, as well as other specific binding factors such as co-factors,or constructed ribozymes or anti-sense RNA molecules of the typesdescribed in section (III.) which can be designed to bind (but designednot to cleave) cancer stem line-specific-intracellular elements.Attachment of fluorescent moieties to monoclonal antibodies for thepurpose of allowing visual detection of bound antibody is a methodcommonly used by those skilled in the art (e.g., immunohistochemistry)and may also be used in conjunction with bound co-factors or constructedribozymes or antisense molecules.

It should be noted that, by the OSES model, the early neoplastic lesion(i.e., collection of symmetrically-dividing stem cells) could in theoryarise as an aberrancy at any point in the development of a given tissue(e.g., during adult tissue genesis or even as early as embryogenesiswhen tissues are being formed). There are indeed some data that theinitiation of certain cancers may occur much earlier than expected byconventional evolutionary models, i.e., during early tissuedevelopment—and thus potentially detectable at these early periods.

For example, there are reports that some young women exposed toradiation at a prepubescent age (prior to mammary gland development)have elevated rates of breast cancer in adulthood (Deng et al, “Loss ofHeterozygosity in Normal Tissue Adjacent to Breast Carcinomas”, Science(Washington D.C.), 274:2057-2059 (1996)). How radiation could becarcinogenic to a tissue which has not yet developed and is not yetproliferating is a concept not adequately explained by conventionalmodels. However, since there is evidence that the normally clonal natureof stem cell micro-environments in developing tissues is a state createdearly in development (Gordon et al, “Differentiation and self-renewal inthe mouse gastrointestinal epithelium”, Cur. Opin. Cell Bio., 6:795-803(1994)), these findings of early radiation-induced breast cancer areconsistent with the OSES model wherein alterations in the initialdevelopment of the gland due to irradiation damage can result in analtered stem cell milieu and thereby predispose to the aberrant birth ofa symmetrically-dividing stem cell (i.e., cancer cell). This populationof symmetrically-dividing stem cells (i.e., cancer cells) may take yearsto emerge in numbers (and thus years to detect by conventional methods)but by the OSES model is, in theory, detectable much earlier.

While the mammary gland is unique in that it undergoes the majority ofits development ex utero, other tissue types develop mostly in uterothereby making detection of aberrant development (i.e., symmetric stemcell mitosis/cancer) in other tissue types possible prior to pubescenceand conceivably possible as early as during embryogenesis.Interestingly, as mentioned previously, there are a group ofindependently reported enigmatic findings that a subset of patientspossess shared genetic alterations (e.g., loss of heterozygosity of WT-1or hypermethylation of H19, loss of heterozygosity of breastcancer-related loci, and micro-satellite mutator phenotype defects) inboth tumorous as well as synchronous non-neoplastic tissues (Chao et al,“Genetic mosaicism in normal tissues of Wilms' tumor patients”, NatureGenet., 3:127-131 (1993); Moulton et al, “Epigenetic lesions at the H19locus in Wilms' tumor patients”, Nature Genet., 7:440-447 (1994). Denget al, “Loss of Heterozygosity in Normal Tissue Adjacent to BreastCarcinomas”, Science (Washington D.C.), 274:2057-2059 (1996); Parsons etal, “Mismatch Repair Deficiency in Phenotypically Normal Human Cells”,Science (Washington D.C.), 268:738-740 (1995)). These findings indicatethat such genomic alterations shared by neoplastic and non-neoplastictissues must have occurred in the common embryonic ancestor cell whichgave rise to both the neoplastic cells as well as normal-appearing cellsof the same tissue type. Conventional models would expect suchmutation-harboring normal tissues (like their neoplastic counterparts),by virtue of their apparent possession of cancer-predisposingalterations, to display histopathological evidence of “overgrowth”—aprediction not supported by the evidence. Such seemingly enigmaticfindings, however, are consistent with the OSES model wherein suchgenomic alterations present in embryonic progenitor cells are passed onto adult stem cells but do not endow a selective advantage, as would beexpected by conventional models, but rather predispose to aberrantdifferentiation of non-stem cells within a stem cell milieu therebypredisposing to subsequent symmetric stem cell mitoses. By this model,in its initial stages, symmetrically-dividing stem cells may not behistologically detectable since their cancer cell progeny aredifferentiating in a relatively orderly manner—however, such a lesionwould be detectable by OSES-derived methods designed todetect-symmetrically-dividing stem cells.

Accordingly, early detection methods arising from the OSES model shouldallow assessment of a cancer prophecy at an early age, at birth, or evenpossibly in utero. Detecting cancer this early would of course not bepossible according to the conventional cancer model which is predicatedupon the notion that cancer involves a series of gradual and cumulativecellular derangements occurring after tissue morphogenesis (i.e., laterin life). Of course, most tumors will not be detectable this early evenby the OSES method, as most tumors will not be initiated until adulthoodas a result of somatic disruption (rather than early developmentaldisruption) of a stem cell milieu. However, adult-onset tumors willalso, for similar reasons, be detectable much earlier by OSES-derivedmethods than by conventional means.

Such an OSES-based method for early cancer detection can be used inpatients at risk for developing cancer (e.g., because of family historyor environmental risks, such as job hazards or smoking) or in patientsin clinical remission from cancer. In addition, perfection of suchtechniques could lead to their widespread utility in routine cancerscreening of the asymptomatic patient.

In order to better illustrate preferred embodiments of the invention,Table 1, Table 2 and Table 3 are provided below which respectivelyidentify gene products involved in asymmetric mitotic pathway stem cellmarkers (SCM's), and asymmetrically acting proteins, RNAs and DNAs.These lists are meant to be exemplary and not exhaustive of genes thatmay be targeted according to the invention. Other gene targets have beenidentified previously, e.g. in section II, subsection 2) of the subjectapplication.

TABLE 1 Gene Products Involved In Asymmetric Mitotic Pathway a) Factorsinvolved in structural asymmetry: notch (mammal) - provides inherentcellular asymmetry via localization at basolateral surface. m-numb(mammal) - homolog of Drosophila numb which is a membrane associatedprotein asymmetrically segregated into daughter neuroblast PTC(mammal) - transmembrane protein, represses Wnt, hh, TGF-beta frizzled(Drosophila) - Wnt receptor smoothened (Drosophila) - Hedgehog receptorinsecutable (Drosophila) - cytoskeletal binding protein, asymmetricallysegregated in neuron development prospero (Drosophila) - involved inasymmetric mitosis Gip-1 (nematode) - cell surface receptor involved inasymmetric determination of cell fate par-1, par-2, par-3, lin-17, andunc-73 (nematode) - asymmetric determination of daughter cell fate skn-1(nematode) - asymmetric determination of daughter cell fate cdc24(yeast) - involved in asymmetric mitosis cdc42 (yeast) - membraneG-protein at previous bud site, asymmetrically segregated to onedaughter cell (has nematode and human homologs) b) Downstreamtranscription factors effecting asymmetric mitosis: Isl-1 (mammal) -asymmetric determination of Islet of Langerhans cell v.s. tubule stemcell in pancreas TTK (Drosophila) - downstream of numb Unc-86(nematode) - equally distributed in both daughter cells, turns on mec-3lin-11 (nematode) - asymmetric determination of vulva epithelium v.shypodermis Sw15 (yeast) - equally distributed in both daughter cells,turns on HO gene. Ash-1p (yeast) - inhibits HO gene c) Factorsultimately responsible for asymmetrically-defining cell type (expressedin 1 of 2 daughter cells): En (Drosophila) - homeobox transcriptionfactor mec-3 (nematode) - transcription factor effecting neurondifferentiation. HO (yeast) - encodes endonuclease, mediates mating typerecombination event¹. Such genes are described in the followingreferences: Hirsch et al, “Pheromone response in yeast”, Bioessays, 14:367-373 (1992); Herman et al, “The Caenorhabditis elegans gene lin-44controls the polarity of asymmetric cell divisions”, Development, 120:1035-1047 (1994); Johnson et al, “Human Homolog of patched, a CandidateGene for the Basal Cell Nevus Syndrome”, Science, 272: 1668-1671 (1996);Way et al, “Cell polarity and the mechanism of asymmetric celldivision”, Bioessays, 16: 925-931 (1994); Lin et al, “Neuroblasts: amodel for the asymmetric division of stem cells”, Trends Genet., 13:33-39 (1997), Perrimon, “Serpentine Proteins Slither into the Winglessand Hedgehog Fields”, Cell, 86: 513-516 (1996), Kuhl et al, “Wntsignalling goes nuclear”, Bioessays, 19: 101-104 (1997); Bowerman et al“The maternal gene skn-1 encodes a protein that is distributed unequallyin early C. elegans embryos”, Cell, 74: 443-452 (1993)). ¹The yeastpheromone response pathway may also include gene products (e.g., cdc28,kar1) whose human homologs are involved in the asymmetric mitoticpathway of stem cells.)

TABLE 2 Stem Cell Markers (SCM's) Tissue Type Stem Cell Markers (SCM/s)Reference Blood hematopoietic stem cell CD34, Scl/Tal-1, Flk-1/KDR, 1,2, 19, 20 Tie-1, Tie-2, c-Kit, AC133 myeloid precursor PU.1 1 lymphoidprecursor ikaros 1 Skin basal stem cell beta-a alpha 2,3,5 integrin, 3cytokeratin19 basonuclin, skin 1a-i/Epoc-1/ 4 Oct 11, LEF-1 Lung basalstem cell cytokeratin 14 5 type II pneumocyte SP-1, SP-2, EGF-R 5 MUC-16 Liver, Pancreas hepatic (oval) stem cell cytokeratin 14, c-Kit, CF 7Ag's 270, 38, 374, 3, 18, 11 8 AFP, IGF-2, TGF-alpha/beta, 8 GGTPancreatic stem cell lsl-1, FA-1  9, 10 Gonads primordial germ cellTRA-1-60, SSEA-1,3,4 11, 12 GI tract intestinal crypt stem cell BCL-213  Breast mammary stem cell Muc-1, ESA 14  Prostate basal prostaticstem cell HMWCk (5, 14), pp32, CD44 15, 16 CNS neural stem cells notch,numb, nestin, p75 17, 18 References for Table 2: 1. Robb et al, TheScl/Tal1 genes: roles in normal and malignant haematopoiesis, Bioessays,19: 607-612 (1997). 2. Robb et al, The hemangioblast - an elusive cellcaptured in culture, Bioessays, 20: 611-614 (1998). 3. Watt, Epidermalstem cells: markers, patterning and the control of stem cell fate, Phil.Trans. Soc. Lond. B, 353: 831-837 (1998). 4. Byrne, Regulation of geneexpression in developing epidermal epithelial, Bioessays, 19: 691-698(1997). 5. Otto, Lung stem cells, Int. J. Exp. Path., 78: 291-310(1997). 6. Jarrad et al, MUC1 is a novel marker for the type IIpneumocyte lineage during lung carcinogenesis, Cancer Res., 58:5582-5589 (1998). 7. Allison et al, Liver stem cells: when the goinggets tough they get going, Int. J. Exp. Path., 78: 365-381 (1997). 8.Sigal et al, The liver as a stem cell and lineage system, Am. J.Physiol., 26: G139-G148 (1992). 9. Way et al, Cell polarity and themechanism of asymmetric cell division, Bioessays, 16: 925-931 (1994).10. Tornehave et al, FA1 immunoreactivity in endocrine tumors and duringdevelopment of human fetal pancreas; negative correlation with glucagonexpression, Histochem. Cell Biol., 106: 535-542 (1996). 11. Andrews,Teratocarcinomas and human embryology: Pluripotent human EC cell lines,APMIS, 106: 158-168 (1998). 12. Shamblott et al, Derivation ofpluripotent stem cells from cultured human primordial germ cells, Proc.Natl. Acad. Sci. USA, 95: 13726-13731 (1998). 13. Potten et al, Theintestinal epithelial stem cell: the mucosal governor, Int. J. Exp.Path., 78: 219-243 (1997). 14. Eaves et al, Phenotypic and functionalcharacterization in vitro of a multipotent epithelial cell present inthe normal adult human breast, Differentiation, 63: 201-13 (1998). 15.Foster et al, Stem cells in prostatic epithelial, Int. J. Exp. Path.,78: 311-329 (1997). 16. Bui, M., et al, Stem cell genes inandrogen-independent prostate cancer, Cancer Metast. Rev., 17: 391-399(1999). 17. Lin et al, Neuroblasts: a model for the asymmetric divisionof stem cells, 13: 33-39 (1997). 18. Anderson, D. J., et al, Celllineage determination and the control of neuronal identity in the neuralcrest, CSHSQB, 62: 493-504 (1997). 19. Ziegler, B. L., et al, KDRreceptor: A key marker defining hematopoietic stem cells, Science, 285:1553-1558 (1999). 20. Yin, A. H., et al AC133, a novel marker for humanhematopoietic stem and progenitor cells, Blood, 90: 5002-5012 (1997).

TABLE 3 References Asymmetrically-Acting Proteins Notch 1-4** 1, 2Pit-1, p78, lsl-1, mPAR-1, MARK 3-7 TFIIIA, Y-box proteins  8piwi/hiwi/elF2C/prg-1/rde-1 homolog**  9, 10 mut-7-homolog 11 X-linkedmodifiers of HD, PWS 12-15 glypican-3 (X-linked modifier of IGF2) 16m-Numb, HASH-1/2, dHAND, BC1 17-19 Tan-1, SCL, Oct-2, spectrosome 20-22hnRNP's (e.g., hnRNP1, A2), snRNP's (e.g., U1-snRNP**)  8, 23 POMp75,POMp100 24 Myo-D, Myf(1, 5) 25 Asymmetrically-acting RNA and/or DNAendogenous sense RNA's: H19 26 SNRPN, 1PW, PAR (1,5 SN), ASR (1,2), BD27-29 ZNF127 (homology with RNP's) 30 aminoacyl tRNA's 43 endogenousanti-sense RNA's: H19, IGF2, IGF2R, KvLQT1 31-33 ZNF127AS, UBE3A 30, 34other interacting RNA and/or DNA: Ins2 35 U2af (homology with U2-snRNP)39, 36 X-linked modifier of X transmission 37 loci 3q21, 3q26, 4qter,16p13, 16q22 38, 39 loci 15p (and centromere), 1 (centromere) 40, 41 DNApairing activity: HPP-1, polypeptides (100 & 75 kDa) 42 U1-snRNP** 23POMp75, POMp100 24 References for Table 3 1. Lendahl, U. A growingfamily of Notch ligands, Bioessays, 20: 103-107 (1998). 2.Artavanis-Tsakonas, S., et al, Notch signaling: Cell fate control andsignal integration in development, Science, 284: 770-776 (1999). 3.Hall, P. A., et al, Stem cells: the generation and maintenance ofcellular diversity, Development, 106: 619-633 (1989). 4. Guo, S., et al,par-1, a gene required for establishing polarity in C. elegans embryos,encodes a putative Ser/Thr kinase that is asymmetrically distributed,Cell, 19: 611-620 (1995). 5. Parsa, I. Loss of a Mr 78,000 marker inchemically induced transplantable carcinomas and primary carcinoma ofthe pancreas, Cancer Res., 48: 2265-2272 (1988). 6. Way et al, Cellpolarity and the mechanism of asymmetric cell division, Bioessays, 16:925-931 (1994). 7. Jan, Y. N., et al, Asymmetric cell division, Nature,392: 775-778 (1998). 8. Ladomery, M. Multifunctional proteins suggestconnection transcriptional and post-transcriptional processes,Bioessays, 19: 903-909 (1997). 9. Benfey, P. N., Stem cells: A tale oftwo kingdoms, Curr. Biol., 9: R171-R172 (1999). 10. Tabara, H., et al,The rde-1 gene, RNA interference, and transposon silencing in C.elegans, Cell, 99: 123-132 (1999). 11. Ketting, R. F., et al, mut-7 ofC. elegans, required for transposon silencing and RNA interference, is ahomolog of Werner Syndrome helicase and RNaseD, Cell, 99: 133-141(1999). 12. Sabl, J. F., et al, Epigene conversion: A proposal withimplications for gene mapping in humans, Am. J. Hum. Genet., 50:1171-1177 (1992). 13. Laird, C. Proposed genetic basis of Huntington

s diseaseö, Trends Genet., 6: 242-247 (1990). 14. Singh, P. B., et al, Asequence motif found in a Drosophila heterochromatin protein isconserved in animals and plants, Nucleic Acids Res., 19: 789-794 (1991).15. Nicholls, R. D. Inciminating gene suspects, Prader-Willi styleö,Nature Genet., 23: 132-134 (1999). 16. Hastie, N. Disomy and diseaseresolved, Nature, 389: 785-786 (1997). 17. Zhong, W. Asymmetriclocalization of a mammalian Numb homolog during mouse corticalneurogenesis, Neuron, 17: 43-53 (1996). 18. Gestblom, C., et al, Thebasic helix-loop-helix transcription factor dHAND, a marker gene for thedeveloping human sympathetic nervous system, is expressed in both high-and low-stage neuroblastomas, Lab. Invest., 79: 67-79 (1999). 19.Miyamoto, T., et al, A SacII polymorphism in the human ASCL2 (HASH2)gene region, J. Hum. Genet., 43: 69-70 (1998). 20. Ellisen, L. W. TAN-1,the human homolog of the Drosophila Notch gene, is broken by chromosomaltranslocations in T lymphoblastic neoplasms, Cell, 66: 649-661 (1991).21. Robb, L., et al, The SCL/TAL1 gene: roles in normal and malignanthematopoiesis, Bioessays, 19: 607-613 (1997). 22. Robb, L., et al, Thehemangioblast - an elusive cell captured in culture, Bioessays, 20:611-614 (1998). 23. Dowjat, K. Anti-(U1)snRNP autoantibodies inhibithomologous pairing activity of the human recombination complex, DNA CellBiol., 16: 819-827 (1997). 24. Bertrand, P. Human POMp75 is identifiedas the pro-oncoprotein TLS/FUS: both POMp75 and POMp100 DNA homologous,pairing activities are associated to cell proliferation, Oncogene, 18:4515-4521 (1999). 25. Braun, T., et al, Myf-5 and myoD genes areactivated in distinct mesenchymal stem cells and determine differentskeletal muscle cell lineage, EMBO J., 15: 10-18 (1996). 26. Schmidt; J.V. et al, Enhancer competition between H19 and Igf2 does not mediatetheir imprinting, Proc. Natl. Acad. Sci. USA, 96: 9733-9738 (1999). 27.Dittrich, B., et al, Imprint switching on human chromosome 15 mayinvolve alternative transcripts of the SNRPN gene, Nat. Genet., 14:163-170 (1996). 28. Barlow, D. Competition - a common motif for theimprinting mechanism, EMBO J., 16: 6899-6905 (1997). 29. Kelsey, G., etal, Imprint switch mechanism indicated by mutations in Prader-Willi andAngelman syndromes, Bioessays, 19: 361-365 (1997). 30. Jong, M. T. C.,et al, A novel imprinted gene, encoding a RING zinc-finger protein, andoverlapping antisense transcript in the Prader-Willi syndrome criticalregion, Hum. Molec. Genet., 8: 783-793 (1999). 31. Moore, T., et al,Multiple imprinted sense and anti-sense transcripts, differentialmethylation and tandem repeats in putative imprinting control regionupstream of mouse Igf2, Proc. Natl. Acad. Sci. USA, 94: 12509-12514(1997). 32. Wutz, A., et al, Imprinted expression of the Igf2r genedepends on an intronic CpG island, Nature, 389: 745-749 (1997). 33.Smilinich, N. J., et al, A maternally methylated CpG island in KvLQT1 isassociated with an antisense paternal transcript and loss of imprintingin Beckwith-Wiedemann syndrome, Proc. Natl. Acad. Sci. USA, 96:8064-8069 (1999). 34. Rougelle, C., et al, An imprinted antisense RNAoverlaps UBE3A and a second maternally expressed transcript, Nat.Genet., 19: 15-16 (1998). 35. Duvillie, B., et al, Imprinting at themouse Ins2 locus: evidence for cis- and trans-allelic interactions,Genomics, 47: 52-57 (1998). 36. Feil, R., et al, Parentalchromosome-specific chromatin conformation in the imprinted U2af1-rs1gene in the mouse, J. Biol. Chem., 272: 20893-20900 (1997). 37. Naumova,A. K., et al, Parental origin-dependent male offspring-specifictransmission ratio distortion at loci on the human X chromosome. Am. J.Hum. Genet., 62: 1493-1499 (1998). 38. Tartof K. D., et al,Trans-sensing effects from Drosophila to humans, Cell, 65: 201-203(1991). 39. Stout, K., et al, Somatic pairing between subtelomericchromosomal regions: implications for human genetic disease, Chrom.Res., 7: 323-329 (1999). 40. Lewis, J. P., et al, Somatic pairing ofcentromeres and short arms of chromosome 15 in the hematopoietic andlymphoid system, Hum. Genet., 92: 577-582 (1993). 41. Arnoldus, P. J.,et al, Somatic pairing of chromosome 1 centromeres in interphase nucleiof human cerebellum, Hum. Genet., 83: 231-234 (1989). 42. Akhmedov, A.T., et al, Characterization of two nuclear mammalian homologousDNA-pairing activities that do not require associated exonucleaseactivity, Proc. Natl. Acad. Sci. USA, 92: 1729-1733 (1995). 43. Jansen,R. -P. RNA - cytoskeletal associations, FASEB J., 13: 455-466 (1999).

SUMMARY

I. Gene Targets that are Identified in the Present Invention

With regard to Section I of Novel Therapies Provided by the Inventionpertaining to immunotherapy directed at stem cell antigens, there areindeed a number of published stem cell antigens. Specifically, stem cellantigens (a.k.a. stem cell markers, a.k.a. cancer stem line-specificmarkers) are known for a number of tissue types (see Table 2) and areavailable as potential targets for therapy. This list (Table 2) is notmeant to be exhaustive, but merely exemplary of stem cell antigens thatmay be targeted in the subject therapies.

With regard to Section II of Novel Therapies Provided by the Inventionpertaining to induction of a switch from symmetric to asymmetric cancerstem line mitosis, there have been a number of published moleculesinvolved in this switch. Specifically, stem cell-specific (or, cancerstem line-specific) molecules involved in the symmetric-asymmetricmitotic switch are known: for a number of tissue types (see, e.g., Table1 and Table 3 and targets identified in Section II) and are available aspotential targets for therapy. Those targets listed in Table 1 and Table3, as well as targets identified in Section II are also meant to beexemplary and not exhaustive of potential targets that may be targetedin this aspect of the invention. Since, as described in Section II, thetherapeutic goal of this aspect of the invention is to force a cancerstem line from symmetric to asymmetric mitosis, these targets (listed inTable-1, Table 2, and Section II of the specification) causing thisswitch would require therapeutic activation whereas those targetsinhibiting this switch will require therapeutic blockage. It should benoted that depending on the tissue type and cellular context some of thelisted targets can at times cause (and at other times inhibit)asymmetric mitosis—thus all asymmetrically-acting species in general(whether seemingly causing or inhibiting asymmetric mitosis) should beconsidered potential targets for therapy and have been listed as such.

With regard to Section III of Novel Therapies Provided by the Inventionpertaining to eradication of a cancer stem line via its induction tosymmetrically differentiate, again there have been a number of publishedcancer stem line-specific molecules involved in this induction.Specifically, as mentioned supra, stem cell-specific (or, cancer stemline-specific) molecules involved in the symmetric-asymmetric mitoticswitch are known for a number of tissue types (see Table 3 and SectionII) and are available as potential targets for therapy. Those targetslisted in Table 3 are again to be considered exemplary and supplementaryto those identified Table 1 and Section II of the subject, application.However as described for the methods of Section III, and incontradistinction to the methods of Section II, the therapeutic goalhere is not to force asymmetric mitosis but rather to maintain a cancerstem line in symmetric mitosis. Such methodology requires the oppositeof what is described in Section II. In other words, those targetscausing a switch to asymmetric mitosis would require therapeuticblockage while those targets inhibiting this switch would requiretherapeutic activation. Again, since there is variability as to thecausative versus inhibitory actions (with respect to induction ofasymmetric mitosis) by the listed targets, depending on the tissue typeand cellular context, it is likely that one target may be causative inone context (and thus worthy of activation by the methods described inSection II) while inhibitory in another context (and thus also worthy ofactivation by the methods described in Section III).

It should be noted that the molecules/gene products listed in Table 3(as well as in Table 1 and Section II) can be generally characterized asspecies of protein, riboprotein; RNA, and DNA which areasymmetrically-acting—and it is this peculiar asymmetrically-actingquality that makes these cancer stem line-specific molecular speciessuch good targets. More specifically, what is meant byasymmetrically-acting is that such molecular species cause asymmetricmitosis and do so because they function as:

1) proteins or riboprotein complexes that

i) unequally segregate to one or another daughter cell (e.g., Notch,Numb, p78) thereby causing differences in cell fate

ii) effect unequal segregation not of themselves but of RNA's todaughter cells

iii) unequally effect the outcome of RNA:RNA interactions (e.g. RNP's,mut-7)

iv) unequally effect the outcome of DNA:DNA interactions (e.g. POM's)

v) unequally effect the fate of daughter of cells via other mechanisms(e.g., piwi, X-linked modifiers)

2) RNA's that

i) unequally effect (imprinted) allele expression (e.g., H19, SNRPN)

ii) act as endogenous anti-sense RNA's to unequally effect (imprinted)allele expression (e.g., UBE3A)

iii) unequally effect the outcome of DNA:DNA interactions (e.g., RNP'S)3) DNA's that

i) are themselves involved in unequal interallelic pairing (e.g., 11p15,15q11-13).

Accordingly, forcing a cancer stem line to switch to an asymmetricmitotic phase (i.e., see next section regarding the goal of Section II)can be accomplished by either therapeutically activatingunequally-acting targets (e.g., those listed in Table 1 and Table 3), orby therapeutically blocking the inhibitors of unequally-acting targets(e.g. those listed in Table 1, and Table 3). Alternatively, forcing acancer stem line to remain in a symmetric mitotic phase (see nextsection regarding the goal of Section III) can be accomplished by eitherblocking unequally-acting targets, or by activating inhibitors ofunequally-acting targets.

II. Updated Use/Design/Construction of Novel Therapeutics

With regard to Section I of Novel Therapies Provided by the Inventionpertaining to immunotherapy directed at stem cell antigens, there isindeed available a number of monoclonal antibodies specific to stem cellmarkers (a.k.a. cancer stem line markers). For example, monoclonalantibodies specific to hematopoietic stem cell markers (i.e., monoclonalantibody to Flk-1/KDR), and to lung stem cell-markers (i.e., monoclonalantibody to SP-A) have been well-described (ref.'s 1-4). Also,therapeutic efficacy has been demonstrated, e.g. in the case ofmonoclonal antibodies to the hematopoietic stem cell marker Flk-1/KDR,albeit in a slightly different context (i.e., as an anti-angiogenic)(ref. 2). Considering that the methods of construction of monoclonalantibodies are well-known by those skilled in the art, additionalmonoclonal antibodies specific to the known cancer stem line-specifictargets (listed in Table 2) can be readily made (with or withoutattached therapeutic, e.g. radionuclide, moieties, as described inSection I) and tested for therapeutic efficacy.

With regard to Section II of Novel Therapies Provided by the Inventionpertaining to induction of a switch from symmetric to asymmetric cancerstem line mitosis, there is indeed available a number of (DNA-, RNA-,antibody, protein, and other molecularly-based) therapeutics that cantarget and dysregulate cancer stem line-specific asymmetrically-actingmolecular species (e.g., those molecular species listed in Table 1,Table 3 and Section II that determine the mitotic phase—i.e., asymmetricversus symmetric). For example, as previously mentioned in Section II:

1) DNA-based therapeutics (i.e., gene therapy) can be used to activategenes that cause asymmetric mitosis—thereby causing a cancer stem lineto switch from symmetric to asymmetric mitosis. Alternatively, DNA-basedtherapeutics (i.e., gene therapy) can be used to activate genes thatblock the inhibition of asymmetric mitosis—thereby causing a cancer stemline to switch from symmetric to asymmetric mitosis.

2) RNA-based therapeutics (e.g., antisense or ribozyme therapy) can beused to block expression of gene products that inhibit asymmetricmitosis—thereby causing a cancer stem line to switch from symmetric toasymmetric mitosis.

Indeed, working published examples of this are the use ofcustom-designed antisense RNA to specifically inhibitasymmetrically-acting piwi homologs (ref. 5), and to inhibit asymmetricRNP action (ref. 6), in both cases resulting in significant cell fatechanges (ref.'s 5, 6). Also, since portions of the 3′ UTR (untranslatedregion) of some RNA's are largely responsible for the asymmetric actionof such RNA's, coupled with the data that these 3′UTR portions containwell-conserved motifs—antisense/ribozyme therapeutics can be readilyconstructed by those skilled in the art, to these motifs so as toinhibit in a general way the unequal action of asymmetrically-acting-RNAspecies—as has been described (ref. 6).

Also not previously mentioned in Section II, but presented here is anupdated version of additional therapeutics (i.e. in addition to thoseDNA- and RNA-based therapies mentioned):

3) RNA-based therapeutics which use endogenous asymmetrically-actingsense RNA species (e.g., H19, SNRPN) to either activate or block theinhibition of asymmetric mitosis—thereby causing a cancer stem line toswitch from symmetric to asymmetric mitosis.

4) RNA-based therapeutics which use endogenous asymmetrically-actingantisense RNA species (e.g., ZNF127AS, UBE3A) to either activate orblock the inhibition of asymmetric mitosis—thereby causing a cancer stemline to switch from symmetric to asymmetric mitosis.

5) Antibody-based therapeutics which block molecular species thatnormally, inhibit asymmetric mitosis—thereby causing a cancer stem lineto switch from symmetric to asymmetric mitosis.

Indeed, a working published example of this is the use of antibodies toU1-snRNP to inhibit inter-allelic interactions occurring via DNA:DNAinteractions (ref. 9).

6) Protein-based therapeutics which use either endogenous or constructedprotein species to either cause asymmetric mitosis or block inhibitionof asymmetric mitosis—thereby causing a cancer stern line to switch fromsymmetric to asymmetric mitosis. For example, certain endogenousinhibitors of Notch (e.g., notch-less) could be used to change themitotic phase of a cancer stem line (i.e., from symmetric to asymmetricmitosis) (ref.'s 7, 8).

Moreover and more generally, since the asymmetric action of certainasymmetrically-acting proteins can be traced to their RNA-bindingaction, coupled with the data that RNA-binding is due to certainwell-conserved protein motifs (e.g., DEAH, or KH protein domains) (ref.6), peptide mimics can be readily constructed by those skilled in theart (e.g., as described for farnesyl transferase inhibitor protein-basedtherapeutics, see ref. 21) for the purpose of therapeutically competingwith (i.e., inhibiting) endogenous asymmetrically-acting protein speciesthereby forcing a cancer stem line to assume an asymmetric mitoticphase. Also, like in the case of farnesyl transferase inhibitor peptides(ref. 21), readily enabled screening assays can be constructed, by thoseskilled in the art, to search for improved (in this case, anti-symmetricmitosis) compounds of protein or other molecular species make-up.

Techniques for more efficient in vivo delivery of RNA-based therapeuticshave been described (e.g., construction of exonuclease-resistant RNAspecies) (ref. 23), as have techniques for more efficient in vivodelivery of protein-based therapeutics (e.g. lipophilic orpeptidase-resistant protein species)(ref.'s 21, 22). Continued technicalimprovements of this sort will enable more efficient in vivo delivery ofthe novel therapeutics described in this application.

In addition to those specific published working examples cited abovethat support the idea that asymmetric mitosis can betherapeutically-induced via delivery of certain DNA-, RNA-, antibody-,and protein-based compounds (e.g. as mentioned, in the cases ofantisense RNA to asymmetrically-acting piwi homologs and RNP-relatedcomplexes, as well as antibodies to RNA's), a number of moregenerally-acting compounds have also been shown to adversely effect themitotic machinery. These include cytoskeletal inhibitors (e.g.,colcemid, colchicine, cytochalasin D, latrunculin A, arsenic and otherheavy metals, taxanes, monastrol) which while not very specific withregard to their molecular targets (as compared to novel DNA-, RNA-,antibody-, and protein-based therapeutics) do still show well-describedanti-cancer activity—albeit with associated toxicities attributable totheir lack of target specificity. Thus these compounds serve as goodcontrols for which to compare newer more specific therapeutic inhibitorsof symmetric mitosis.

Also, a number of generally-acting differentiation/starvation-inducingcompounds are known to have anti-cancer activity through their action onthe differentiation/asymmetric mitosis pathway—some of these compoundsinclude, but are not exclusive to, retinoic acid enzymes involved innucleic: acid (DNA or RNA) synthesis, protein synthesis, the removal ofessential growth factors, the use of drugs, or the use of chalones thatinduce a cellular starvation response, histone deacetylase inhibitors(e.g. trichostatin), sodium phenylbutyrate, sodium phenylacetate, DMSO,HMBA, PMA, tetramethyl urea, amino acid analogs (e.g. AzC, 6MMPR,L-alanosine, PALA), inosine, monophosphate dehydrogenase inhibitors(e.g., mycophenolic acid), methotrexate, rRNA inhibitors (e.g., heparin,synthetic peptide substrate of casein kinase II, actinomycin D,puromycin aminonucleoside, DRB, H1o histone), inhibitors of chargingtRNA or protein translation (e.g., histidinol, EIF46 cleavage), guaninenucleotide inhibitors (e.g., virazole, 6-chloropurine), anddifferentiation-inducing ligands/receptor pathway components (e.g.,Wnt/frizzled and downstream components, Hedgehog/Patched and downstreamcomponents, Notch/Delta/Serrate and downstream components). It should benoted that these compounds can effect both differentiation/starvation aswell as more downstream events involving the asymmetric mitoticmachinery—thus can also (like the mentioned cytoskeletal inhibitors)serve as good controls for which to compare newer more specifictherapeutic-inhibitors of symmetric mitosis.

With regard to section III of Novel Therapies Provided by the Inventionpertaining to induction of symmetric differentiation in the cancer stemline, i.e., by 1) inhibition of asymmetric cancer stem line mitosis,followed by 2) induction of cancer stem line differentiation) there isindeed available as mentioned a number of (DNA-, RNA-, antibody, andprotein-based) therapeutics that can target cancer stem line-specificasymmetrically-acting molecular species are listed in Table 1, and Table3 and Section II of this application). Also to be included astherapeutics that alter the mitotic program and/ordifferentiation/starvation state of a cancer stem line are thepreviously mentioned cytoskeletal inhibitors (e.g., colcemid, et al) anddifferentiation/starvation inducers (e.g., retinoic acid, et al). Itshould be noted that the differentiation/starvation inducers (e.g.,retinoic acid, et al) may have overlapping roles as both 1) dysregulatorof mitosis, and 2) inducer of differentiation, and thus some of thesecompounds may be used for both of these processes.

It should also be aptly noted that, unlike for Section II where thetherapeutic goal is to induce asymmetric mitosis (e.g., by eitheractivating factors that cause asymmetric mitosis, or blocking factorsthat inhibit asymmetric mitosis), the therapeutic goal of Section III isthe opposite—i.e., to either block factors that cause a switch fromsymmetric to asymmetric mitosis, or activate factors that inhibitasymmetric mitosis. In this way (i.e., the therapeutic goal outlined inSection III):

1) a cancer stem line will be forced to assume a symmetric mitoticprogram (versus the therapeutic goal outlined in Section II which is toforce a cancer stem line to assume an asymmetric mitotic program). Thiscan be accomplished crudely by cytoskeletal inhibitors (e.g., colcemid,et al) and differentiation/starvation inducers (e.g., retinoic acid, etal), or specifically by the described novel DNA-, RNA-, antibody-,protein, and other molecularly-based therapies.

2) after the cancer stem line is induced to remain in a symmetricmitotic program, the final therapeutic goal (as outlined in Section III)is to cause it to undergo a differentiation/starvation program—whichwill be symmetric in nature since the cancer stem line has beentherapeutically frozen in a symmetric mitotic program. This can beaccomplished most efficiently by differentiation/starvation inducers(e.g., retinoic acid, et al), but may also be effected by cytoskeletalinhibitors e.g., colcemid, et al) or the described novel DNA-, RNA-,antibody-, protein, and other molecularly-based therapies.

There are indeed data supportive of these ideas. Namely, as outlined inSection III, the therapeutic goal is a two-step one: 1) inhibit (cancerstem line) asymmetric mitosis, and then 2) induce (cancer stein line)differentiation. There are indeed published examples, albeitpreliminary, of a related 2-step therapeutic design whereby cancer cellsare 1) affected at the level of their mitotic machinery, and 2) affectedvia differentiation/starvation. These examples provide scientificevidence in support of the premise of the efficacy of the therapeuticmethods of the invention. For example, cancer cells have been shown torespond to the following therapeutic combinations:

Agent effecting mitotic Agent effecting Therapeutic phasedifferentiation/starvation Effect Reference Arsenic retinoic aciddifferentiation 10 and apoptosis Arsenic GM-CSF differentiation 11activation c-myc sodium phenylbutyrate apoptosis 20 activation c-mycserum starvation apoptosis 12 activation c-myc p53 apoptosis 13inhibition p53 differentiation 14, 15 GTP-tubulin E7 HMBA apoptosis 16p130 (Rb-like) cytokine-deprivation differentiation 17 loss of Rbmitogen-deprivation apoptosis 18 loss senescence serum deprivationapoptosis 19

That c-myc activates apoptosis in the context of a cellular conflict(i.e. opposing signals of mitosis and differentiation/starvation) isindeed an increasingly well-appreciated concept, and was a topic of arecent review (ref. 24).

Accordingly, these systems which have been already been shown capable ofdifferentiation/apoptosis (in response to relatively crude methods), canserve as controls for which to test (and optimize) the novel DNA-, RNA-,antibody-, protein-, and other molecularly-based therapies described inthis application.

REFERENCES CITED IN SUMMARY

-   1. Ziegler, B. L., et al, “KDR receptor: A key maker defining    hematopoietic stem cells,” Science, 285: 1553-1558 (1999).-   2. Witte, L., et al, “Monoclonal antibodies targeting the VEGF    receptor-2 (Flk1/KDR) as an anti-angiogenic therapeutic strategy,”    Cancer Metast. Rev., 17: 155-161 (1998).-   3. Ten Have-Opbroek, A. A. W., et al, “The alveolar type II cell is    a pluripotential stem cell in the genesis of human adenocarcinomas    and squamous cell carcinomas”, Histol. Histopathol., 12: 319-336    (1997).-   4. SP-1 monoclonal antibody is commercially available from Byk    Gulden Co. (Lomberg. Chemische Fabrik GmbH, D-78467. Konstanz)    Germany.-   5. Benfey, P. N., “Stem cells: A tale of two kingdoms,” Curr. Biol.,    9:R171-R172 (1999).-   6. King, M. L., et al, “Polarizing genetic information in the egg:    RNA localization in the frog oocyte,” Bioessays, 21: 546-557 (1999).-   7. Panin, V. M, et al, “Modulators of Notch signaling,” Semin. Cell    Biol., 9: 609-617 (1998).-   8. Royet, J., et al, “Notchless encodes a novel    WD40-repeat-containing protein that modulates Notch signaling    activity,” EMBO J., 17:7351-7360 (1998).-   9. Dowjat, K. “Anti-(U1)snRNP autoantibodies inhibit homologous    pairing activity of the human recombination complex,” DNA Cell    Biol., 16: 819-827 (199.7).-   10. Soignet, S. L., et al, “Complete remission after treatment of    acute promyelocytic leukemia with arsenic trioxide,” N. Eng. J.    Med., 339:1341-1348 (1998).-   11. Kizaki, M., et al, “Application of heavy metal and cytokine for    differentiation-inducing therapy in acute promyelocytic    leukemia,” J. Natl. Cancer Inst., 90: 1906-1907 (1998).-   12. Evan, G. I., et al, “Induction of apoptosis in fibroblasts by    c-mycprotein,”Cell, 69: 119-128 (1992).-   13. Hermeking, H., et al, “Mediation of c-myc-induced apoptosis by    p53,” Science, 265: 2091-2093 (1994).-   14. Li, Y. M., et al, “Arsenic targets tubulins to induce apoptosis    in myeloid leukemia cells”, Cancer Res., 59: 776-780 (1999).-   15. Sherley, J. L., et al, “ôExpression of the wild-type p53    antioncogene induces guanine nucleotide-dependent stem cell division    kinetics”, Proc. Natl. Acad. Sci. USA, 92: 136-140 (1995).-   16. Richon, V. M., et al, “Two cytodifferentiation agent-induced    pathways, differentiation and apoptosis, are distinguished by the    expression of human papillomavirus 16 E7 in human bladder carcinoma    cells,” Cancer Res., 57:2789-2798 (1997).-   17. Mori, A., et al, “Granulocytic differentiation of myeloid    progenitor cells by p130, the retinoblastoma tumor suppressor    homologue,” Oncogene, 18: 6209-6221 (1999).-   18. Wang, J., et al, “Rb functions to inhibit apoptosis during    myocyte differentiation”, Cancer Res., 57: 351-354 (1997).-   19. Preston, G. A., et al, “Regulation of apoptosis by low serum in    cells of different stages of neoplastic progression: enhanced    susceptibility after loss of a senescence gene and decreased    susceptibility after loss of a tumor suppressor gene,” Cancer Res.,    54: 4214-4223 (1994).-   20. Bernhard, D., et al, “Apoptosis induced by the histone    deacetylase inhibitor sodium butyrate in human leukemic    lymphoblasts,” FASEB J., 13:1991-2001 (1999).-   21. Lobell, R. B., et al, “Pre-clinical development of    farnesyltransferase inhibitors,” Cancer Metast. Rev., 17: 203-210    (1998).-   22. Schumacher, T. N. M, et al, “Identification of D-peptide ligands    through mirror-image phage display,” Science, 271: 1854-1857 (1996).-   23. Teplova; M., et al, “Structural origins of the exonuclease    resistance of a zwitterionic RNA,” Proc. Natl. Acad. Sci. USA, 96:    14240-14245 (1999).-   24. Fuhrmann, G., et al, “The MYC dualism in growth and death,”    Mutation Res., 437: 205-217 (1999).

The invention may be embodied in other specific forms without departingfrom its spirit or essential characteristics. The described embodimentsare to be considered in all respects only as illustrative and notrestrictive, and the scope of the invention is, therefore, indicated bythe appended claims rather than by the foregoing description. Allmodifications which come within the meaning and range of the lawfulequivalency of the claims are to be embraced within that scope.

1. A method of treating cancer comprising administering to a humanpatient diagnosed with cancer an antibody that binds to AC133 in anamount sufficient to inhibit the proliferation of cancer cells in thepatient, wherein the cancer is not acute leukemia.
 2. A method oftreating cancer comprising administering to a human patient diagnosedwith cancer an antibody fragment attached to a therapeutic moiety,wherein said antibody fragment binds to AC133, in an amount sufficientto inhibit the proliferation of cancer cells in the patient, wherein thecancer is not acute leukemia.
 3. A method of treating cancer comprisingadministering to a human patient diagnosed with cancer an antibody thatbinds to AC133 in an amount sufficient to inhibit the proliferation ofcancer cells in the patient, wherein the cancer is not leukemia.
 4. Amethod of treating cancer comprising administering to a human patientdiagnosed with cancer an antibody fragment attached to a therapeuticmoiety, wherein said antibody fragment binds to AC133, in an amountsufficient to inhibit the proliferation of cancer cells in the patient,wherein the cancer is not leukemia.
 5. A method of treating cancercomprising administering to a human patient diagnosed with a solid tumoran antibody that binds to AC133 in an amount sufficient to inhibit theproliferation of cancer cells in the patient.
 6. A method of treatingcancer comprising administering to a human patient diagnosed with asolid tumor an antibody fragment attached to a therapeutic moiety,wherein said antibody fragment binds to AC133, in an amount sufficientto inhibit the proliferation of cancer cells in the patient.
 7. Themethod of claim 1, 2, 3, 4, 5, or 6, wherein the cancer cells arereduced in the patient.
 8. The method of claim 1, 2, 3, 4, 5, or 6,wherein the cancer cells do not increase in the patient.
 9. The methodof claim 7, wherein the method further comprises detecting cancer cells.10. The method of claim 9, wherein the method detects a reduction intumor size.
 11. The method of claim 9, wherein the detection utilizes aspecimen from the patient.
 12. The method of claim 11, wherein thespecimen is from a blood sample, a bone marrow sample, or a tumorbiopsy.
 13. The method of claim 9, wherein the detection utilizes animaging technique.
 14. The method of claim 13, wherein the technique isaccomplished using radionuclide imaging, fluorescent imaging, CT scan,X-ray or MRI scan.
 15. The method of claim 8, wherein the method furthercomprises detecting cancer cells.
 16. The method of claim 15, whereinthe method detects that tumor size does not increase.
 17. The method ofclaim 15, wherein the detection utilizes a specimen from the patient.18. The method of claim 17, wherein the specimen is from a blood sample,a bone marrow sample, or a tumor biopsy.
 19. The method of claim 15,wherein the detection utilizes an imaging technique.
 20. The method ofclaim 19, wherein the technique is accomplished using radionuclideimaging, fluorescent imaging, CT scan, X-ray or MRI scan.
 21. The methodof claim 1, 2, 3, 4, 5, or 6, wherein the method further comprisesdetecting cancer cells.
 22. The method of claim 21, wherein cancer cellsdo not increase.
 23. The method of claim 21, wherein the method detectsa reduction in cancer cells.
 24. The method of claim 21, wherein thedetection utilizes a specimen from the patient.
 25. The method of claim24, wherein the specimen is from a blood sample, a bone marrow sample,or a tumor biopsy.
 26. The method of claim 21, wherein the detectionutilizes an imaging technique.
 27. The method of claim 26, wherein thetechnique is accomplished using radionuclide imaging, fluorescentimaging, CT scan, X-ray or MRI scan.
 28. The method of claim 1, 2, 3, 4,5, or 6, wherein the method further comprises detecting cancer stemcells.
 29. The method of claim 28, wherein the detection utilizes aspecimen from the patient.
 30. The method of claim 29, wherein thespecimen is from a blood sample, a bone marrow sample, or a tumorbiopsy.
 31. The method of claim 28, wherein the detection utilizes animaging technique.
 32. The method of claim 28, wherein the technique isaccomplished using radionuclide imaging, fluorescent imaging, CT scan,X-ray or MRI scan.
 33. The method of claim 1, 3, or 5, wherein theantibody is attached to a therapeutic moiety.
 34. The method of claim33, wherein the therapeutic moiety is selected from alkylating agents,anti-metabolites, plant alkaloids, chemotherapeutic agents,radionuclides, therapeutic enzymes, cytokines, cytotoxins, or growthmodulators.
 35. The method of claim 1, 2, 3, 4, 5, or 6, wherein theantibody or fragment thereof results in a decrease in viability ofcancer stem cells.
 36. The method of claim 1, 2, 3, 4, 5, or 6, whereinthe antibody or fragment thereof results in a decrease in growth ofcancer stem cells.
 37. The method of claim 1, 2, 3, 4, 5, or 6, whereinthe antibody or fragment thereof modulates cancer stem cells.
 38. Themethod of claim 1, 2, 3, 4, 5, or 6, wherein the antibody or fragmentthereof results in the differentiation of cancer stem cells.
 39. Themethod of claim 1, 2, 3, 4, 5, or 6, wherein the antibody or fragmentthereof results in asymmetric cell division of cancer stem cells. 40.The method of claim 1, 2, 3, 4, 5, or 6, wherein the antibody orfragment thereof binds to the target that is expressed on a cancer stemcell.
 41. The method of claim 1, 2, 3, 4, 5, or 6, wherein the patienthas been diagnosed with a solid tumor and has undergone cancer therapy.42. The method of claim 1, 2, 3, 4, 5, or 6, wherein the patient hasbeen diagnosed with a hematologic cancer and has undergone cancertherapy.
 43. The method of claim 1, 2, 3, 4, 5, or 6, wherein thepatient has undergone cancer therapy.
 44. The method of claim 43,wherein the patient is in remission from cancer.
 45. The method of claim43, wherein the patient has relapsed from cancer.
 46. The method ofclaim 43, wherein the patient has failed cancer treatment.
 47. Themethod of claim 1, 2, 3, 4, 5, or 6, wherein the cancer is brain cancer,neural cancer, breast cancer, kidney cancer, colon cancer, prostatecancer, rhabdomyosarcoma, retinoblastoma, liver cancer, skin cancer,gastrointestinal cancer, pancreatic cancer, cervical cancer, or lungcancer.
 48. The method of claim 37, wherein the cancer stem cells to bemodulated are slow growing.
 49. The method of claim 37, wherein thecancer stem cells to be modulated are mutationally spared relative totumor bulk.
 50. The method of claim 37, wherein the cancer stem cells tobe modulated symmetrically divide.
 51. The method of claim 2, 4, or 6,wherein the therapeutic moiety is selected from alkylating agents,anti-metabolites, plant alkaloids, chemotherapeutic agents,radionuclides, therapeutic enzymes, cytokines, cytotoxins, or growthmodulators.
 52. The method of claim 1, 2, 3, 4, 5, or 6, wherein theantibody or fragment thereof kills cancer stem cells when assayed invitro.
 53. The method of claim 1, 2, 3, 4, 5, or 6, wherein the antibodyor fragment thereof inhibits the proliferation of cancer stem cells whenassayed in vitro.
 54. The method of claim 1, 2, 3, 4, 5, or 6, whereinthe antibody or fragment thereof results in a decrease in viability ofcancer stem cells when assayed in vitro.
 55. The method of claim 1, 2,3, 4, 5, or 6, wherein the antibody or fragment thereof results in adecrease in growth of cancer stem cells when assayed in vitro.
 56. Themethod of claim 1, 2, 3, 4, 5, or 6, wherein the antibody or fragmentthereof modulates cancer stem cells when assayed in vitro.
 57. Themethod of claim 1, 2, 3, 4, 5, or 6, wherein the antibody or fragmentthereof kills cancer stem cells.
 58. The method of claim 1, 2, 3, 4, 5,or 6, wherein the antibody or fragment thereof inhibits theproliferation of cancer stem cells.
 59. The method of claim 1, 2, 3, 4,5, or 6, wherein the method comprises the initial therapy for treatingthe patient.